Vaccination in newborns and infants

ABSTRACT

The present invention relates to vaccines comprising at least one mRNA encoding at least one antigen for use in the treatment of a disease in newborns and/or infants, preferably exhibiting an age of not more than 2 years, preferably of not more than 1 year, more preferably of not more than 9 months or even 6 months, wherein the treatment comprises vaccination of the newborn or infant and eliciting an immune response in said newborn or infant. The present invention is furthermore directed to kits and kits of parts comprising such a vaccine and/or its components and to methods applying such a vaccine or kit.

This application is a continuation of U.S. application Ser. No.16/273,525, filed Feb. 12, 2019, which is a continuation of U.S.application Ser. No. 16/179,990, filed Nov. 4, 2018, now U.S. Pat. No.10,596,252, which is a continuation of U.S. application Ser. No.15/466,308, filed Mar. 22, 2017, now U.S. Pat. No. 10,172,935, which isa continuation of U.S. application Ser. No. 13/824,705, filed Jul. 10,2013, now U.S. Pat. No. 9,623,095, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2012/000877, filed Feb. 29, 2012, which claims benefit ofInternational Application No. PCT/EP2011/001047, filed Mar. 2, 2011, theentire contents of each of which are hereby incorporated by reference.

The present invention relates to vaccines comprising at least one mRNAencoding at least one antigen for use in the treatment of a disease innewborns and/or infants, preferably exhibiting an age of not more than 2years, preferably of not more than 1 year, more preferably of not morethan 9 months or even 6 months, wherein the treatment comprisesvaccination of the newborn or infant and eliciting an immune response insaid newborn or infant. The present invention is furthermore directed tokits and kits of parts comprising such a vaccine and/or its componentsand to methods applying such a vaccine or kit.

Diagnosing, preventing and treating infections and allergies in newbornsand infants are of major interest and increasingly the subject ofintense research worldwide. In this context, a profound knowledge aboutthe mechanisms of the immune system of newborns and infants is of majorimportance. As widely known, the primary role of the immune system is toprotect the organism against pathogens, but the response of the immunesystem to such pathogens is not equal throughout the whole life span. Itis further known that responses of the immune system undergoage-associated alterations. Likewise, responses of the immune system ofnewborns and infants are not equal to those of adults. Particularly theresponse of T and B cells differs in many aspects, a fact, which may becontributed to the necessity of the prenatal requirements of the fetalimmune system and the transition to the external conditions duringbirth.

As is well known, all organ systems of the body undergo a dramatictransition during birth from a sheltered intra-uterine existence to theradically distinct environment of the outside world. This acutetransition is then followed by a gradual, age-dependent maturation. Asreviewed by Ofer (see Ofer, NATURE REVIEWS|IMMUNOLOGY VOLUME 7|MAY2007|379) the fetal and neonatal immune systems are usually associatedwith physiological demands that are three-fold: protection againstinfection, including viral and bacterial pathogens at the maternal-fetalinterface, avoidance of potentially harmful pro-inflammatory/T helper 1(Th1)-cell polarizing responses that can induce alloimmune reactionsbetween mother and fetus, and mediation of the transition between thenormally sterile intra-uterine environment to the foreign antigen-richenvironment of the outside world, including primary colonization of theskin and intestinal tract by microorganisms. Given the limited exposureto antigens in utero and the well-described defects in neonatal adaptiveimmunity, newborns must rely on their innate immune systems forprotection to a significant extent. As the innate immune system caninstruct the adaptive immune response, distinct functional expression ofneonatal innate immunity, including a bias against Th1-cell-polarizingcytokines, contributes to a distinct pattern of neonatal adaptive immuneresponses. Mounting evidence indicates that infection-induced productionof pro-inflammatory/Th1 cell-polarizing cytokines, includingtumour-necrosis factor (TNF) and interleukin-1β (IL-1β), is associatedwith premature labour and pre-term delivery. In particular, TNFproduction is thought to favour abortion through the induction ofapoptosis in placental and fetal cells. The ability of pro-inflammatorycytokines to induce spontaneous abortion is likely to be an importantreason for the strong bias of the maternal and fetal immune systems ofmultiple mammalian species towards TH2-cell-polarizing cytokines (seeLevy, 2007, supra).

Because of this impaired production of Th1 cell associated cytokines, itwas initially thought that the neonatal innate immune system wasgenerally impaired or depressed; however, stimulus-induced production ofcertain cytokines (for example, IL-6, IL-10 and IL-23) by neonatalmonocytes and antigen presenting cells (APCs) actually exceeds that ofadults (see Angelone, D. et al., Pediatr. Res. 60, 205-209 (2006);Vanden Eijnden, S., Goriely, S., De Wit, D., Goldman, M. & Willems, F.Eur. J. Immunol. 36, 21-26, (2006); Chelvarajan, R. L. et al., J.Leukoc. Biol. 75, 982-994 (2004)). Nevertheless, there still appears tobe a bias against Th1 cell-polarizing cytokines, which leaves thenewborn susceptible to microbial infections and contributes to theimpairment of neonatal immune responses to most vaccines, therebyfrustrating efforts to protect this vulnerable population. After birth,there is an age-dependent maturation of the immune response. Of note,prenatal and postnatal exposure to environmental microbial products thatcan activate innate immunity might accelerate this maturation process,particularly if the exposure occurs repeatedly over time, diminishingTH2-cell polarization and/or enhancing Th1 cell polarization and therebypotentially reducing allergy and atopy, in accord with the hygienehypothesis (see again Levy, 2007, supra).

T cell mediated immune responses in early life, particularly innewborns, have been reviewed by Marchant and Goldmann (see Marchant andGoldmann, Clinical and Experimental Immunology, 2005, 14: 10-18). Asstated there circulating neonatal T lymphocytes are fundamentallydifferent from naïve adult T cells and have characteristics of recentthymic emigrants. They contain high concentrations of T cell-receptorexcision circles (TRECs), episomal DNA by products of TCRα-chainrearrangement that are not replicated but diluted during cell division.Like adult naïve cells, most neonatal T lymphocytes express the CD45 RA⁺isoform and the costimulatory molecules CD27 and CD28. In contrast toadult naïve lymphocytes, neonatal lymphocytes express the CD38 molecule.In addition, a high proportion of circulating neonatal T cells are incycle and display an increased susceptibility to apoptosis indicatinghigh cell turn-over. Proliferation of naïve T lymphocytes can also bedetected during fetal life and could last up to five years of age. Thehigh cell turn-over observed in early life probably plays a central rolein the establishment of the T cell repertoire. Despite their highturn-over, T cells preserve long telomeric sequences through a highconstitutive telomerase activity. In vitro apoptosis of neonatal Tlymphocytes can be prevented by cytokines signalling through the γ-chainof the IL-2 receptor, namely IL-2, IL-4, IL-7 and IL-15. Among thesecytokines, IL-7 and IL-15 also induce the proliferation of neonatal Tlymphocytes in the absence of other stimuli. IL-7 is involved inthymocyte development at a stage preceding the T cell receptorrearrangement. IL-15 preferentially stimulates the proliferation of CD8rather than CD4+ T cells. In contrast to IL-7, IL-15 induces thedifferentiation of CD8+ T lymphocytes in vitro (see Marchant andGoldmann, 2005, supra).

Several mechanisms limit T helper 1 (Th1) type responses in early life.In utero, Th1 responses are toxic to the placenta and are inhibited bytrophoblast-derived IL-10 and progesterone. At birth, Th1 responses arestill of lower magnitude than later in life. In vitro, CD4+ T cells ofnewborns produce lower levels of IFNγ than naïve T cells of adults andare hypermethylated at CpG and non-CpG sites within the IFNγ promoter.In the presence of suboptimal CD28 costimulation, IL-12 stimulates theproduction of both IL-4 and IFNγ by neonatal CD4 T lymphocytes whereasadult cells do not produce IL-4 under similar conditions. In response topolyclonal or superantigen activation, postnatal thymocytes develop intoTh2 cytokine producing CD4 cells whereas IL-12 is required to stimulatethe production of IFNγ. In contrast, neonatal CD8+ T cells producesimilar levels of IFNγ and have a pattern of IFNγ promoter methylationcomparable to that of naïve adult cells. In addition, neonatal CD8 Tlymphocytes are strictly dependent on the presence of IL-4 at the timeof priming to differentiate into IL-4-producing cells. The capacity ofneonatal CD4 T lymphocytes to express CD40 ligand (CD154), a moleculeplaying a critical role in the help to B lymphocytes and CD8+ T cells,remains controversial (see also Marchant and Goldmann, 2005, supra).

Together, the available data indicate that naïve T lymphocytes aredifferently programmed in neonates and in adults. As may be summarized,the capacity of neonatal CD4+ T cells to produce IFNγ and of neonatalDCs to promote Th1 responses is lower as compared to adults in in vitrostudies. Additionally, Th1 responses to a number of vaccines andinfectious pathogens in vivo are poor during early life. However, matureTh1 responses can develop in certain conditions such as neonatal BCGvaccination and Bordetella pertussis infection, probably in relationwith a more efficient activation of DCs. Accordingly, the classicalparadigm that newborns have incompetent T lymphocytes developing onlyweak or even tolerogenic responses has to be reconsidered. Theobservation that mature cellular immune responses can be developed inearly life suggests that under appropriate conditions of stimulationneonatal T lymphocytes may be instructed to fight intracellularpathogens (see also Marchant and Goldmann, 2005, supra).

Not only differences in T cell responses but also differences in B cellresponses appear to exhibit a severe impact on immunoprotection innewborns and/or infants with respect to adult individuals. It has longbeen thought that the vulnerability of children younger than 18-24months of age to encapsulated bacteria such as pneumococcus, Haemophilusinfluenzae B (Hib) and meningococcus has long been thought to reflect ageneral failure to generate T cell-independent B cell responses to mostbacterial polysaccharides (see Claire-Anne Siegrist* and RichardAspinall, NATURE REVIEWS|IMMUNOLOGY VOLUME 9|MARCH 2009|185). However,immaturity of the immune system in newborns and/or infants also has adirect effect on the magnitude of antibody responses to T cell-dependentprotein antigens. Mechanisms that shape B cell responses in early lifewere investigated using neonatal mouse immunization models that weredeveloped to reproduce the main limitations of immune responses tovaccines that are administered at an early age (see Claire-AnneSiegrist* and Richard Aspinall, 2009, supra). There are numerousdifferences between neonatal and adult mouse splenic B cells, althoughfewer differences have been identified by comparing human peripheral Bcells. Specifically, human neonatal B cells express lower levels of theco-stimulatory molecules CD40, CD80 and CD86, which decreases theirresponses to CD40 ligand (CD40L) and interleukin-10 (IL-10) expressed byT cells. Splenic marginal zone infant B cells express lower levels ofCD21, which limits their capacity to respond topolysaccharide-complement complexes. The expression of TACI(transmembrane activator and calcium-modulating cyclophilin-ligandinteractor; also known as TNFRSF13B), an important co-stimulatoryreceptor, is also decreased on both neonatal mouse and neonatal human Bcells, particularly those born prematurely. In addition, B cellresponses in early life are influenced by numerous extrinsic factors.Antibodies of maternal origin bind to vaccine antigens in an epitopespecific manner and therefore prevent infant B cells from accessingimmunodominant vaccine epitopes. Furthermore, human and mouse neonateshave low levels of serum complement component C3, which limits theirresponses to antigen-C3d complexes. The human spleen contains fewermarginal zone macrophages (which have a crucial role in the induction ofan antibody response through the trapping of particulate antigens) inneonates than in adults, and the cells differ in their capacity toproduce cytokines. In infant mice, B cell responses are limited by amarked delay in the maturation of the follicular dendritic cell (FDC)network, resulting from the failure of FDC precursors to respond to Bcell-mediated lymphotoxin-α signalling. FDCs nucleate germinal centrereactions by attracting antigen-specific B cells, retain antigens in theform of immune complexes that are highly stimulatory to B cells andprovide signals that lead to somatic hypermutation and class-switchrecombination. The immaturity of the FDC network therefore delays theinduction and limits the magnitude of germinal centre responses, evenwhen potent adjuvants that induce adult-like B cell, T cell and DCactivation patterns are used. Additional to the postnatal maturation ofantibody responses, the antibody persistence in vivo exhibits animportant effect. The long-term maintenance of specific antibodies witha short half-life requires the persistence of antibody-producing Bcells, which can either be continually produced from a pool of memory Bcells or can persist as long-lived plasma cells. Antibody mediateddepletion of memory B cells, which does not affect plasma cells, hasshown that the plasma-cell stage is independent of the memory B cellpool. It has also been shown, that persistence of antibodies in vivo maybe influenced by environmental factors. This supports the hypothesisthat the limited persistence of antibody responses in early life resultsfrom exposure to a large load of environmental antigens, which resultsin competition for access to a limited set of plasma-cell survivalniches in the bone marrow (see Claire-Anne Siegrist* and RichardAspinall, 2009, supra).

As known before, the administration of a single dose of vaccine at birthcan fail to elicit specific antibodies while priming for subsequentsecondary responses, which indicates a preferential neonataldifferentiation pathway towards memory B cells rather than plasma cells.Several factors seem to contribute to this B cell differentiationpattern. The fate of antigen-specific naive B cells and theirdifferentiation to short-lived plasma cells, long-lived plasma cells ormemory B cells is controlled by early B cell activation signals.High-affinity B cells are actively recruited to the plasma-cell pool,whereas moderate-affinity B cells remain as memory B cells in thesecondary lymphoid organs. So, decreased initial B cell receptoraffinity and/or delayed affinity maturation of neonatal naive B cellsmight decrease the strength of the signal and favour memory B celldifferentiation.

The limited expression of CD21 by infant B cells also supports thegeneration of memory B cells and impairs the development of plasmacells, which would be supported by CD40-mediated signalling, cytokinessuch as 11-21 and interactions with ligands such as B cell activatingfactor (BAFF; also known as TNFSF13B) and APRIL. Remarkably, theseplasma-cell-supporting factors are all expressed at low levels early inlife, unless additional activation signals are provided to enhance DCand T cell activation. Furthermore, early-life B cells might have tocompete for limited resources within the germinal centre, which impairsantibody responses. Plasma-cell differentiation may thus be ‘forced’ inearly life by providing additional DC activation signals. Therefore, acombination of factors appears to result in the preferentialdifferentiation of early-life B cells towards memory B cells instead oflong-lived plasma cells in a pattern. Importantly, although it has beenshown that the memory B cell pool can be formed early in life, thisshould not be considered as evidence that its magnitude or persistenceis similar to that elicited in immunologically mature hosts. Recentobservations of the failure of booster vaccines to elicit a memoryresponse in adolescents or young adults that had been primed againsthepatitis B virus in infancy suggest that infant-triggered memory Bcells might not last life long. Whether this reflects a smaller B cellpool in infants and/or the influence of as yet undefined homeostaticfactors remains to be determined. In summary, a large number of B cellintrinsic factors and extrinsic determinants appear to cooperate tolimit the induction and the persistence of antibody-secreting plasmacells in early life, while supporting the preferential induction ofmemory B cell responses (see Claire-Anne Siegrist* and Richard Aspinall,2009, supra).

Alterations in T cell responses and also in B cell responses asdiscussed above exhibit a significant effect not only onimmunoprotection towards pathogens but also to vaccination strategies innewborns and/or infants when combating infectious diseases but possiblyalso allergies, autoimmune disorders or further diseases. Many attemptshave been carried out to provide efficient vaccines, which may overcomeat least some of the above limitations.

One previous approach to overcome theses deficiencies in the context ofvirus-based influenza vaccines refers to the administration of naked DNAplasmid (pHA) expressing hemagglutinin (HA) from the neurovirulentstrain A/WSN/33 of influenza virus to prime protective immune responsesby inoculating newborn and adult mice (see Bot et al, 1997,International Immunology, Vol. 9, No. 11, pp. 1641-1650). As shown byBot et al. (1997) continuous exposure to small doses of antigensubsequent to neonatal DNA immunization may lead to priming of specificB and Th cells, rather than tolerance induction. However, only pHAimmunization of adult mice primed a strongly biased Th1 response,whereas in neonates it induced a mixed Th1/Th2 response. One furthervery similar approach of the same working group was directed to thecombined administration of plasmids expressing nucleoprotein (NP) orhemagglutinin (HA) of influenza virus. Neonatal immunization of BALB/cmice was followed by priming of B cells, Th cells and CTL rather thantolerance (see Bot et al., Vaccine, Vol. 16, No. 17, pp. 16751682,1998). However, protection in terms of survival against lethal challengewith homologous or heterologous strains was not reported to be complete.Further, in the case of NP expressing plasmid, the protective immunityelicited by neonatal immunization required a longer time to develop, ascompared with adult immunization. Neither Bot et al. (1997, supra) norBot et al. (1998, supra) showed good Th1 responses in neonates.Furthermore, even though it was stressed in both papers that DNAvaccination represents an efficient and safe means to generate broadhumoral and cellular immune responses to influenza viruses during theearliest stages of postnatal life, DNA has been encountered meanwhile asdangerous due to unwanted insertion into the genome. Such DNA basedvaccinations may even lead to interruption of functional genes andcancer or the formation of anti-DNA antibodies and are therefore out offocus as of today.

Further approaches were directed to improvement of delivery systems andadministration of immunomodulators to optimize vaccine responses inearly life. Jiri Kovarik and Claire-Anne Siegrist focus on problemsarising from the attempt to vaccinate against pathogens very early inlife and on the role of selective adjuvants that could be used to: (i)rapidly induce strong antibody responses of the appropriate isotypes;(ii) elicit sustained antibody responses extending beyond infancy; (iii)induce efficient Th1 and CTL responses in spite of the preferential Th2polarization of early life responses; (iv) escape from maternal antibodymediated inhibition of vaccine responses; (v) show acceptablereactogenicity in early life; and (vi) allow incorporation of severalvaccine antigens into a single formulation so as to reduce the number ofrequired injections (see Jiri Kovarik and Claire-Anne Siegrist,Immunology and Cell Biology (1998) 76, 222-236). Kovarik and Siegrist(1998, supra) inter alia discuss different antigen delivery systems suchas administration of particulate substances, emulsions, liposomes,virosomes, microspheres, live vaccines, vectors and DNA vaccines as wellas the use of immunomodulators such as MPL, QS21, MDP derivatives,cytokines, interferons, and CpG oligodesoxynucleotides and combinationsof antigen presentation systems and immunomodulators. However, aslikewise shown in Kovarik and Siegrist (1998, supra) many of thesecombinations are hypothetical and may not even provide an efficient Th1response or even lead to unwanted side effects.

Similarly, Adrian Bot and Constantin Bona (see Adrian Bot and ConstantinBona, Microbes and Infection 4 (2002) 511-520) suggest the use ofbacterial CpG motifs to activate immature antigen-presenting cells andto enhance neonatal immunogenicity of DNA vaccines. Additionally, Botand Bona (2002, supra) suggest a combination with subsequent boostingusing conventional vaccines. Nevertheless, the strategy outlined in thispaper does not lead to convincing Th1 responses. Furthermore, theapproach is based on the use of DNA vaccine, which may be regarded aspotentially dangerous as outlined above.

A further promising but very specific strategy relies on the use of thespecific novel adjuvant IC31. As known in the art, there are only fewadjuvants approved for human use. One major adjuvant approved for humanuse is e.g. Alum, an aluminium salt based adjuvant. However, althoughapproved for human use, such aluminium salts failed to providesatisfactory augmentation of immune responses for seasonal influenzavaccines in early human clinical trials, an effect, which may beexpected likewise during other vaccination strategies. Further licensedadjuvanted influenza vaccines include to date FLUAD® (NovartisVaccines), containing MF59 in combination with a subunit vaccineformulation, and the virosomal vaccines INFLEXAL®V (Berna Biotech, aCrucell company) and INVIVAC® (Solvay). Although animal studies andhuman clinical trials revealed a higher immunogenicity profile—definedas increased antibody responses—with the MF59-adjuvanted influenzavaccine, MF59 is not a potent adjuvant for the induction of type 1driven cellular immune responses. Unlike FLUAD®, the virosomal vaccinesrepresent reconstituted influenza virus envelopes containing thefunctional influenza surface proteins haemagglutinin and neuraminidasein their phospholipid bilayer. The immunogenicity and local tolerabilityof virosome-based influenza vaccines has been shown in several studies.However, the development of virosomal formulations is very complex andthe costs of goods are high.

In this context, Kamath et al. (see Kamath et al., 2008, PLoS ONE 3(11):e3683. doi:10.1371/journal.pone.0003683) report the use of a specificadjuvant IC31 with Ag85bESAT-6 fusion protein for immunization ofneonatal mice and adult mice. Conversely to Alum, IC31H induced strongTh1 and Th17 responses in both age groups, characterized bymultifunctional T cells expressing IL-2 and TNFα with or without IFNγ.In the draining lymph nodes, a similarly small number of DC containedthe adjuvant and/or the antigen following neonatal or adultimmunization. Expression of CD40, CD80, CD86 and IL-12p40 production wasfocused on the minute adjuvant-bearing DC population, wherein DCtargeting/activation was similar in adults and neonates. These DC/T cellresponses resulted in an equivalent reduction of bacterial growthfollowing infection with M. bovis BCG, whereas no protection wasobserved when Alum was used as adjuvant. However, no further adjuvantsare shown in Kamath et al. (2008, supra), which allow extension of thisspecific example to other vaccines.

Summarizing the above, none of the present prior art vaccines allow toefficiently evoke immune responses in newborns and/or infants, whichshow at least similar characteristics as an immune response in adults.Particularly, many vaccines fail to provide an efficient Th1 immuneresponse in newborns and/or infants. Accordingly, there is an urgentneed for vaccines optimized for such patients. More precisely, vaccinesare required, which do not bear the problems shown in the prior art orat least diminish these problems to a significant extent. Furthermore,it is highly envisaged to provide vaccines, which allow inducing Th1immune responses in newborns and/or infants, preferably without leadingto a shift from Th1 to Th2 immune responses subsequent toadministration. Likewise, the administration of DNA based vaccinesshould be avoided due to possible insertion of DNA into the genome,possible interruption of genes and formation of anti-DNA antibodies.

The object underlying the present invention is solved by the subjectmatter of the attached claims, more preferably as outlined in thefollowing.

According to a first embodiment, the object underlying the presentinvention is solved by a vaccine comprising at least one mRNA encodingat least one antigen for use in the prophylaxis and/or treatment of adisease in newborns and/or infants, preferably exhibiting an age of notmore than 2 years, preferably of not more than 1 year, more preferablyof not more than 9 months or even 6 months, wherein the treatmentcomprises vaccination of the newborn or young infant and eliciting animmune response in said newborn or infant.

Without being bound to theory RNA vaccines elegantly integrateadjuvanticity and antigen expression, thereby mimicking relevant aspectsof viral infections. This increases their efficacy compared to otherinactivated (dead) vaccines that require the use of advanced adjuvantsin a newborn or an infant, simplifying handling and production. RNA canaddress a range of dedicated immunologic pattern recognition receptors,including toll-like receptors 3, 7, and 8, RIG-I, MDA5, PKR, and othersthat may act synergistically and serve to enhance the induction ofantigen-specific adaptive B and T cell responses. Importantly, byantigen synthesis in transfected host cells, mRNA vaccines directlyintroduce antigen into cellular antigen processing and presentationpathways, granting access to MHC molecules and triggering T cellresponses, irrespective of the hosts MHC haplotype. This enables theinduction of polyclonal T cell responses that may act synergisticallywith other immune responses, including B cells. Also, presenting thefull spectrum of MHC-binding epitopes may circumvent limitations byimmature immune systems in a newborn or an infant. Also, endogenousproduction of antigen ensures faithful posttranslational modification(e.g. proteolytic processing, glycosylation, etc.) that may positivelyimpact immunogenicity. Also, RNA vaccines exhibit safety features thatmake them superior for use in newborns and/or infants. For example, theincreased reactogenicity of live attenuated vaccines generally preventsuse in this highly relevant target group. However, considering the shortpersistence and traceless decay of the vaccine vector within a matter ofdays the observed good immunogenicity is unexpected and contrasts claimsfor plasmid DNA vaccines that variously linked efficacy to thepersistent expression of antigen.

The at least one mRNA of the inventive vaccine as defined in the firstembodiment of the present invention, encoding at least one antigen, maybe selected from any antigen, known to a skilled person, preferablysuitable to elicit an antigen-specific immune response in a patient.According to the present invention, the term “antigen” refers to asubstance which is recognized by the immune system and is capable oftriggering an antigen-specific immune response, e.g. by formation ofantibodies or antigen-specific T cells as part of an adaptive immuneresponse. In this context, the first step of an adaptive immune responseis the activation of naïve antigen-specific T cells or different immunecells able to induce an antigen-specific immune response byantigen-presenting cells. This occurs in the lymphoid tissues and organsthrough which naïve T cells are constantly passing. The three cell typesthat can serve as antigen-presenting cells are dendritic cells,macrophages, and B cells. Each of these cells has a distinct function ineliciting immune responses. Tissue dendritic cells take up antigens byphagocytosis and macropinocytosis and are stimulated by contact withe.g. a foreign antigen to migrate to the local lymphoid tissue, wherethey differentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents or other appropriate stimuli to express MHC molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may also be important to induce T cells. Presenting theantigen on MHC molecules leads to activation of T cells which inducestheir proliferation and differentiation into armed effector T cells. Themost important function of effector T cells is the killing of infectedcells by CD8+ cytotoxic T cells and the activation of macrophages by Th1cells which together make up cell-mediated immunity, and the activationof B cells by both TH2 and Th1 cells to produce different classes ofantibody, thus driving the humoral immune response. T cells recognize anantigen by their T cell receptors which do not recognize and bindantigen directly, but instead recognize short peptide fragments e.g. ofpathogens' protein antigens, which are bound to MHC molecules on thesurfaces of other cells.

In the context of the present invention, antigens as encoded by the atleast one mRNA of the inventive vaccine typically comprise any antigen,falling under the above definition, more preferably protein and peptideantigens. In accordance with the invention, antigens as encoded by theat least one mRNA of the inventive vaccine may be antigens generatedoutside the cell, more typically antigens not derived from the hostorganism (e.g. a human) itself (i.e. non-self antigens) but ratherderived from host cells outside the host organism, e.g. pathogenicantigens, particularly viral antigens, bacterial antigens, fungalantigens, protozoological antigens, animal antigens (preferably selectedfrom animals or organisms as disclosed herein), allergy antigens, etc.Antigens as encoded by the at least one mRNA of the inventive vaccinemay be furthermore antigens generated inside the cell, the tissue or thebody, e.g. by secretion of proteins, their degradation, metabolism, etc.Such antigens include antigens derived from the host organism (e.g. ahuman) itself, e.g. tumour antigens, self-antigens or auto-antigens,such as auto-immune self-antigens, etc., but also (non-self) antigens asdefined above, which have been originally been derived from host cellsoutside the host organism, but which are fragmented or degraded insidethe body, tissue or cell, e.g. by (protease) degradation, metabolism,etc.

Pathogenic antigens particularly comprise e.g. antigens from influenza,preferably influenza A, influenza B, influenza C or thogotovirus,preferably influenza antigens haemagglutinin (HA) and/or neuraminidase(NA), preferably influenza antigens derived from haemagglutinin subtypesH1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14 or H15,and/or neuraminidase subtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9, orpreferably selected from influenza A subtypes H1N1, H1N2, H2N2, H2N3,H3N1, H3N2, H3N3, H5N1, H5N2, H7N7 or H9N2, or any further combination,or from matrix protein 1 (M1), ion channel protein M2 (M2),nucleoprotein (NP), etc; or e.g. antigens from respiratory syncytialvirus (RSV), including F-protein, G-protein, etc.

One further class of antigens as encoded by the at least one mRNA of theinventive vaccine comprises allergy antigens. Allergy antigens aretypically antigens, which cause an allergy in a human and may be derivedfrom either a human or other sources. Such allergy antigens may beselected from antigens derived from different sources, e.g. fromanimals, plants, fungi, bacteria, etc. Allergens in this context alsoinclude antigens derived from e.g. grasses, pollens, molds, drugs, ornumerous environmental triggers, etc. Allergy antigens typically belongto different classes of compounds, such as proteins or peptides andtheir fragments, carbohydrates, polysaccharides, sugars, lipids,phospholipids, etc. Of particular interest in the context of the presentinvention are antigens, which are encoded by the at least one mRNA ofthe inventive vaccine, i.e. protein or peptide antigens and theirfragments or epitopes, or nucleic acids and their fragments,particularly nucleic acids and their fragments, encoding such protein orpeptide antigens and their fragments or epitopes.

Particularly preferred, antigens derived from animals, which are encodedby the at least one mRNA of the inventive vaccine, may include antigensderived from, without being limited thereto, insects, such as mite (e.g.house dust mites), mosquito, bee (e.g. honey bee, bumble bee),cockroach, tick, moth (e.g. silk moth), midge, bug, flea, wasp,caterpillar, fruit fly, migratory locust, grasshopper, ant aphide, fromcrustaceans, such as shrimps, crab, hill, lobster, prawn, crawfish,scampi, from birds, such as duck, goose, seagull, turkey, ostrich,chicken, from fishes, such as eel, herring, carp, seabream, codfish,halibut, catfish, beluga, salmon, flounder, mackerel, cuttlefish, perch,form molluscs, such as scallop, octopus, abalone, snail, whelk, squid,clam, mussel, from spiders, from mammals, such as cow, rabbit, sheep,lion, jaguar, leopard, rat, pig, buffalo, dog, loris, hamster, guineapig, fallow deer, horse, cat, mouse, ocelot, serval, from arthropod,such as spider, or silverfish, from worms, such as nematodes, fromtrichinella species, or roundworm, from amphibians, such as frogs, orfrom sea squirt, etc. Antigens derived from animals may also compriseantigens contained in animal products, preferably contained in animalproducts derived from animals as defined above, e.g. milk, eggs, meat,etc., but also from excrements or precipitates of any kind, derived fromany of these animals.

Most preferably, antigens derived from animals, which are encoded by theat least one mRNA of the inventive vaccine, may include antigens derivedfrom such animals, causing a disease as defined herein, preferably aninfectious disease or an autoimmune disease as defined herein, or anyfurther disease as defined herein.

Antigens derived from plants, which are encoded by the at least one mRNAof the inventive vaccine, may include antigens derived from, withoutbeing limited thereto, fruits, such as kiwi, pineapple, jackfruit,papaya, lemon, orange, mandarin, melon, sharon fruit, strawberry,lychee, apple, cherry paradise apple, mango, passion fruit, plum,apricot, nectarine, pear, passion fruit, raspberry, grape, fromvegetables, such as garlic, onion, leek, soya bean, celery, cauliflower,turnip, paprika, chickpea, fennel, zucchini, cucumber, carrot, yam,bean, pea, olive, tomato, potato, lentil, lettuce, avocado, parsley,horseradish, chirimoya, beet, pumpkin, spinach, from spices, such asmustard, coriander, saffron, pepper, aniseed, from crop, such as oat,buckwheat, barley, rice, wheat, maize, rapeseed, sesame, from nuts, suchas cashew, walnut, butternut, pistachio, almond, hazelnut, peanut,brazil nut, pecan, chestnut, from trees, such as alder, hornbeam, cedar,birch, hazel, beech, ash, privet, oak, plane tree, cypress, palm, fromflowers, such as ragweed, carnation, forsythia, sunflower, lupine,chamomile, lilac, passion flower, from grasses, such as quack grass,common bent, brome grass, Bermuda grass, sweet vernal grass, rye grass,or from other plants, such as opium poppy, pellitory, ribwort, tobacco,asparagus, mugwort, cress, etc.

Antigens derived from fungi, which are encoded by the at least one mRNAof the inventive vaccine, may include antigens derived from, withoutbeing limited thereto, e.g. Alternia sp., Aspergillus sp., Beauveriasp., Candida sp., Cladosporium sp., Endothia sp., Curcularia sp.,Embellisia sp., Epicoccum sp., Fusarium sp., Malassezia sp., Penicillumsp., Pleospora sp., Saccharomyces sp., etc.

Antigens derived from bacteria, which are encoded by the at least onemRNA of the inventive vaccine, may include antigens derived from,without being limited thereto, e.g. Bacillus tetani, Staphylococcusaureus, Streptomyces griseus, etc.

One further class of antigens as encoded by the at least one mRNA of theinventive vaccine comprises tumour antigens. “Tumour antigens” arepreferably located on the surface of the (tumour) cell. Tumour antigensmay also be selected from proteins, which are overexpressed in tumourcells compared to a normal cell. Furthermore, tumour antigens alsoinclude antigens expressed in cells which are (were) not themselves (ororiginally not themselves) degenerated but are associated with thesupposed tumour. Antigens which are connected with tumour-supplyingvessels or (re)formation thereof, in particular those antigens which areassociated with neovascularization, e.g. growth factors, such as VEGF,bFGF etc., are also included herein. Antigens connected with a tumourfurthermore include antigens from cells or tissues, typically embeddingthe tumour. Further, some substances (usually proteins or peptides) areexpressed in patients suffering (knowingly or not-knowingly) from acancer disease and they occur in increased concentrations in the bodyfluids of said patients. These substances are also referred to as“tumour antigens”, however they are not antigens in the stringentmeaning of an immune response inducing substance. The class of tumourantigens can be divided further into tumour-specific antigens (TSAs) andtumour-associated-antigens (TAAs). TSAs can only be presented by tumourcells and never by normal “healthy” cells. They typically result from atumour specific mutation. TAAs, which are more common, are usuallypresented by both tumour and healthy cells. These antigens arerecognized and the antigen-presenting cell can be destroyed by cytotoxicT cells. Additionally, tumour antigens can also occur on the surface ofthe tumour in the form of, e.g., a mutated receptor. In this case, theycan be recognized by antibodies. According to the invention, the terms“cancer diseases” and “tumour diseases” are used synonymously herein.

Examples of tumour antigens as encoded by the at least one mRNA of theinventive vaccine may comprise e.g. antigens selected from the groupcomprising, without being limited thereto, 5T4, 707-AP (707 alanineproline), 9D7, AFP (alpha-fetoprotein), AlbZIP HPG1,alpha5beta1-Integrin, alpha5beta6-Integrin, alpha-methylacyl-coenzyme Aracemase, ART-4 (adenocarcinoma antigen recognized by T cells 4), B7H4,BAGE-1 (B antigen), BCL-2, BING-4, CA 15-3/CA 27-29, CA 19-9, CA 72-4,CA125, calreticulin, CAMEL (CTL-recognized antigen on melanoma), CASP-8(caspase-8), cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CD30,CD33, CD40, CD52, CD55, CD56, CD80, CEA (carcinoembryonic antigen),CLCA2 (calcium-activated chloride channel-2), CML28, Coactosin-likeprotein, Collagen XXIII, COX-2, CT9/BRD6 (bromodomain testis-specificprotein), Cten (C-terminal tensin-like protein), cyclin B 1, cyclin D1,cyp-B (cyclophilin B), CYPB1 (cytochrom P450 1B1), DAM-10/MAGE-B1(differentiation antigen melanoma 10), DAM-6/MAGE-B2 (differentiationantigen melanoma 6), EGFR/Her1, EMMPRIN (tumour cell-associatedextracellular matrix metalloproteinase inducer/), EpCam (epithelial celladhesion molecule), EphA2 (ephrin type-A receptor 2), EphA3 (ephrintype-A receptor 3), ErbB3, EZH2 (enhancer of Zeste homolog 2), FGF-5(fibroblast growth factor-5), FN (fibronectin), Fra-1 (Fos-relatedantigen-1), G250/CAIX (glycoprotein 250), GAGE-1 (G antigen 1), GAGE-2(G antigen 2), GAGE-3 (G antigen 3), GAGE-4 (G antigen 4), GAGE-5 (Gantigen 5), GAGE-6 (G antigen 6), GAGE-7b (G antigen 7b), GAGE-8 (Gantigen 8), GDEP (gene differentially expressed in prostate), GnT-V(N-acetylglucosaminyltransferase V), gp100 (glycoprotein 100 kDa), GPC3(glypican 3), HAGE (helicase antigen), HAST-2 (human signet ringtumour-2), hepsin, Her2/neu/ErbB2 (human epidermalreceptor-2/neurological), HERV-K-MEL, HNE (human neutrophil elastase),homeobox NKX 3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HST-2,hTERT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IGF-1R, IL-13Ra2 (interleukin 13 receptor alpha 2 chain),IL-2R, IL-5, immature laminin receptor, kallikrein 2, kallikrein 4,Ki67, KIAA0205, KK-LC-1 (Kita-kyushu lung cancer antigen 1), KM-HN-1,LAGE-1 (L antigen), livin, MAGE-A1 (melanoma antigen-A1), MAGE-A10(melanoma antigen-A10), MAGE-A12 (melanoma antigen-A12), MAGE-A2(melanoma antigen-A2), MAGE-A3 (melanoma antigen-A3), MAGE-A4 (melanomaantigen-A4), MAGE-A6 (melanoma antigen-A6), MAGE-A9(melanoma-antigen-A9), MAGE-B 1 (melanoma-antigen-B1), MAGE-B10(melanoma-antigen-B 10), MAGE-B 16 (melanoma-antigen-B 16), MAGE-B 17(melanoma-antigen-B 17), MAGE-B2 (melanoma-antigen-B2), MAGE-B 3(melanoma-antigen-B3), MAGE-B 4 (melanoma-antigen-B4), MAGE-B5(melanoma-antigen-B 5), MAGE-B 6 (melanoma-antigen-B 6), MAGE-C1(melanoma-antigen-C1), MAGE-C2 (melanoma-antigen-C2), MAGE-C3(melanoma-antigen-C3), MAGE-D1 (melanoma-antigen-D1), MAGE-D2(melanoma-antigen-D2), MAGE-D4 (melanoma-antigen-D4), MAGE-E1(melanoma-antigen-E1), MAGE-E2 (melanoma-antigen-E2), MAGE-F1(melanoma-antigen-F1), MAGE-H1 (melanoma-antigen-H1), MAGEL2 (MAGE-like2), mammaglobin A, MART-1/Melan-A (melanoma antigen recognized by Tcells-1/melanoma antigen A), MART-2 (melanoma antigen recognized by Tcells-2), matrix protein 22, MC1R (melanocortin 1 receptor), M-CSF(macrophage colony-stimulating factor gene), mesothelin, MG50/PXDN, MMP11 (M-phase phosphoprotein 11), MN/CA IX-antigen, MRP-3 (multidrugresistance-associated protein 3), MUC1 (mucin 1), MUC2 (mucin 2), NA88-A(NA cDNA clone of patient M88), N-acetylglucos-aminyltransferase-V,Neo-PAP (Neopoly(A) polymerase), NGEP, NMP22, NPM/ALK(nucleophosmin/anaplastic lymphoma kinase fusion protein), NSE(neuron-specific enolase), NY-ESO-1 (New York esophageous 1), NYESO-B,OA1 (ocular albinism type 1 protein), OFA-iLRP (oncofetalantigen-immature laminin receptor), OGT (O-linked N-acetylglucosaminetransferase gene), OS-9, osteocalcin, osteopontin, p15 (protein 15),p15, p190 minor bcr-abl, p53, PAGE-4 (prostate GAGE-like protein-4),PAI-1 (plasminogen acitvator inhibitor 1), PAI-2 (plasminogen acitvatorinhibitor 2), PAP (prostate acic phosphatase), PART-1, PATE, PDEF,Pim-1-Kinase, Pin1 (Propyl isomerase), POTE, PRAME (preferentiallyexpressed antigen of melanoma), prostein, proteinase-3, PSA(prostate-specific antigen), PSCA, PSGR, PSM, PSMA (prostate-specificmembrane antigen), RAGE-1 (renal antigen), RHAMM/CD168 (receptor forhyaluronic acid mediated motility), RU1 (renal ubiquitous 1), RU2 (renalubiquitous 1), S-100, SAGE (sarcoma antigen), SART-1 (squamous antigenrejecting tumour 1), SART-2 (squamous antigen rejecting tumour 1),SART-3 (squamous antigen rejecting tumour 1), SCC (squamous cellcarcinoma antigen), Sp17 (sperm protein 17), SSX-1 (synovial sarcoma Xbreakpoint 1), SSX-2/HOM-MEL-40 (synovial sarcoma X breakpoint), SSX-4(synovial sarcoma X breakpoint 4), STAMP-1, STEAP (six transmembraneepithelial antigen prostate), surviving, survivin-2B (intron 2-retainingsurvivin), TA-90, TAG-72, TARP, TGFb (TGFbeta), TGFbRII (TGFbetareceptor II), TGM-4 (prostate-specific transglutaminase), TRAG-3 (taxolresistant associated protein 3), TRG (testin-related gene), TRP-1(tyrosine related protein 1), TRP-2/6b (TRP-2/novel exon 6b), TRP-2/INT2(TRP-2/intron 2), Trpp8, Tyrosinase, UPA (urokinase-type plasminogenactivator), VEGF (vascular endothelial growth factor), VEGFR-2/FLK-1(vascular endothelial growth factor receptor-2), WT1 (Wilm' tumourgene), or may comprise e.g. mutant antigens expressed in cancer diseasesselected from the group comprising, without being limited thereto,alpha-actinin-4/m, ARTC1/m, bcr/abl (breakpoint cluster region-Abelsonfusion protein), beta-Catenin/m (beta-Catenin), BRCA1/m, BRCA2/m,CASP-5/m, CASP-8/m, CDC27/m (cell-division-cycle 27), CDK4/m(cyclin-dependent kinase 4), CDKN2A/m, CML66, COA-1/m, DEK-CAN (fusionprotein), EFTUD2/m, ELF2/m (Elongation factor 2), ETV6-AML1 (Ets variantgene6/acute myeloid leukemia 1 gene fusion protein), FN1/m (fibronectin1), GPNMB/m, HLA-A*0201-R170I (arginine to isoleucine exchange atresidue 170 of the alpha-helix of the alpha2-domain in the HLA-A2 gene),HLA-A11/m, HLA-A2/m, HSP70-2M (heat shock protein 70-2 mutated),KIAA0205/m, K-Ras/m, LDLR-FUT (LDR-Fucosyltransferase fusion protein),MART2/m, ME1/m, MUM-1/m (melanoma ubiquitous mutated 1), MUM-2/m(melanoma ubiquitous mutated 2), MUM-3/m (melanoma ubiquitous mutated3), Myosin class I/m, neo-PAP/m, NFYC/m, N-Ras/m, OGT/m, OS-9/m, p53/m,Pml/RARa (promyelocytic leukemia/retinoic acid receptor alpha), PRDX5/m,PTPRK/m (receptor-type protein-tyrosine phosphatase kappa), RBAF600/m,SIRT2/m, SYTSSX-1 (synaptotagmin I/synovial sarcoma X fusion protein),SYT-SSX-2 (synaptotagmin I/synovial sarcoma X fusion protein), TEL-AML1(translocation Ets-family leukemia/acute myeloid leukemia 1 fusionprotein), TGFbRII (TGFbeta receptor II), TPI/m (triosephosphateisomerase). According to a specific aspect, however, mRNAs encodingantigens gp100, MAGE-A1, MAGE-A3, MART-1/melan-A, survivin, and/ortyrosinase, more preferably mRNAs encoding antigens gp100, MAGE-A1,MAGE-A3, MART-1/melan-A, survivin, and/or tyrosinase, wherein the mRNAshave been complexed with or stabilized with protamine (e.g. in a ratioof about 80 μg mRNA and 128 μg protamine), may be excluded from thescope of invention. In a preferred aspect the tumour antigens as encodedby the at least one mRNA of the inventive vaccine are selected from thegroup consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m,alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1,BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsinB, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD40, CD52, CD55,CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66,COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m,EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2,FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin,Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin classI/m, NA88-A, Nacetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF,Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin,survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1,TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b,TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, and WT1.

According to a particularly preferred aspect, tumour antigens as encodedby the at least one mRNA of the inventive vaccine are selected from thegroup consisting of MAGE-A1 (e.g. MAGE-A1 according to accession numberM77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accessionnumber NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according toaccession number NM_005511), GP100 (e.g. GP100 according to accessionnumber M77348), tyrosinase (e.g. tyrosinase according to accessionnumber NM_000372), survivin (e.g. survivin according to accession numberAF077350), CEA (e.g. CEA according to accession number NM_004363),Her-2/neu (e.g. Her-2/neu according to accession number M11730), WT1(e.g. WT1 according to accession number NM_000378), PRAME (e.g. PRAMEaccording to accession number NM_006115), EGFRI (epidermal growth factorreceptor 1) (e.g. EGFRI (epidermal growth factor receptor 1) accordingto accession number AF288738), MUC1, mucin-1 (e.g. mucin-1 according toaccession number NM_002456), SEC61G (e.g. SEC61G according to accessionnumber NM_014302), hTERT (e.g. hTERT accession number NM_198253), 5T4(e.g. 5T4 according to accession number NM_006670), NY-Eso-1 (e.g.NY-Eso-1 according to accession number NM_001327), TRP-2 (e.g. TRP-2according to accession number NM_001922), STEAP, PCA, PSA, PSMA, etc.

Particularly preferred, antigens are selected from Influenza A virus(HA, NA, NP, M2, M1 antigens), influenza B virus (HA, NA antigens),respiratory syncytial virus (F, G, M, SH antigens), parainfluenza virus(glycoprotein antigens), Streptococcus pneumoniae (pPht, PcsB, StkPantigens), Corynebacterium diphtheriae, Clostridium tetani, Measles,Mumps, Rubella, Rabies virus (G, N antigens), Staphylococcus aureus(toxin antigen), Clostridium difficile (toxin antigen), Mycobacteriumtuberculosis (acute and dormant antigens), Candida albicans, Haemophilusinfluenzae B (HiB), poliovirus, hepatitis B virus (surface and coreantigens), human papillomavirus (L1, L2, E6, E7), human immunodeficiencyvirus (gp120, gag, env antigens), SARS CoV (spike protein),Staphylococcus aureus (IsdA, IsdB, toxin antigens), Pertussis toxin,polio virus (VP1-4), Plasmodium (NANP, CSP protein, ssp2, ama1, msp142antigens), Staphylococcus aureus (IsdA, IsdB, toxin), Bordetellapertussis (toxin), polio virus VP1-4, Plasmodium (NANP, CSP protein,ssp2, ama1, msp142 antigens)

Antigens as encoded by the at least one mRNA of the inventive vaccinemay furthermore comprise fragments of such antigens as mentioned herein,particularly of protein or peptide antigens. Fragments of such antigensin the context of the present invention may comprise fragmentspreferably having a length of about 6 to about 20 or even more aminoacids, e.g. fragments as processed and presented by MHC class Imolecules, preferably having a length of about 8 to about 10 aminoacids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragmentsas processed and presented by MHC class II molecules, preferably havinga length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18,19, 20 or even more amino acids, wherein these fragments may be selectedfrom any part of the amino acid sequence. These fragments are typicallyrecognized by T cells in form of a complex consisting of the peptidefragment and an MHC molecule, i.e. the fragments are typically notrecognized in their native form.

Fragments of antigens as defined herein may also comprise epitopes ofthose antigens. Epitopes (also called “antigen determinants”) aretypically fragments located on the outer surface of (native) protein orpeptide antigens as defined herein, preferably having 5 to 15 aminoacids, more preferably having 5 to 12 amino acids, even more preferablyhaving 6 to 9 amino acids, which may be recognized by antibodies, i.e.in their native form.

According to a further particularly preferred aspect, the tumourantigens as encoded by at least one mRNA of the inventive vaccine mayform a cocktail of antigens, e.g. in an active (immunostimulatory)composition or a kit of parts (wherein preferably each antigen iscontained in one part of the kit), preferably for eliciting an(adaptive) immune response for the treatment of a disease or disorder asdefined herein. For this purpose, the inventive vaccine may comprise atleast one mRNA, wherein each mRNA may encode at least one, preferablytwo, three, four or even more (preferably different) antigens asmentioned herein. Alternatively, the inventive vaccine may contain atleast one, two, three, four or even more (preferably different) mRNAs,wherein each mRNA encodes at least one antigen as mentioned herein.

Such a cocktail of antigens, as encoded by the least one mRNA of theinventive vaccine may be used e.g. in the treatment of e.g. prostatecancer (PCa), preferably in the treatment of neoadjuvant and/orhormone-refractory prostate cancers, and diseases or disorders relatedthereto. For this purpose, the inventive vaccine may comprise at leastone mRNA, wherein each mRNA may encode at least one, preferably two,three, four or even more (preferably different) antigens as mentionedherein. Alternatively, the inventive vaccine may contain at least one,two, three, four or even more (preferably different) mRNAs, wherein eachmRNA encodes at least one antigen as mentioned herein. Preferably, theantigens are selected from PSA (Prostate-Specific Antigen)=KLK3(Kallikrein-3), PSMA (Prostate-Specific Membrane Antigen), PSCA(Prostate Stem Cell Antigen), and/or STEAP (Six Transmembrane EpithelialAntigen of the Prostate).

Furthermore, such a cocktail of antigens, as encoded by the at least onemRNA of the inventive vaccine may be used in the treatment of e.g.non-small cell lung cancers (NSCLC), preferably selected from the threemain sub-types squamous cell lung carcinoma, adenocarcinoma and largecell lung carcinoma, or of disorders related thereto. For this purpose,the inventive vaccine may comprise at least one mRNA, wherein each mRNAmay encode at least one, preferably two, three, four, five, six, seven,eight, nine, ten eleven or twelve (preferably different) antigens asmentioned herein. Alternatively, the inventive vaccine may contain atleast one, preferably two, three, four, five, six, seven, eight, nine,ten, eleven or twelve (preferably different) mRNAs, wherein each mRNAencodes at least one antigen as mentioned herein. Preferably, suchantigens are selected from hTERT, WT1, MAGE-A2, 5T4, MAGE-A3, MUC1,Her-2/neu, NY-ESO-1, CEA, Survivin, MAGE-C1, and/or MAGE-C2.

In the above aspects, each of the above defined antigens may be encodedby one (monocistronic) mRNA. In other words, in this case the at leastone mRNA of the inventive vaccine may comprise at least two (three,four, etc.) (monocistronic) mRNAs, wherein each of these at least two(three, four, etc.) (monocistronic) mRNAs may encode, e.g. just one(preferably different) antigen, preferably selected from one of theabove mentioned antigen combinations.

According to a particularly preferred aspect, the at least one mRNA ofthe inventive vaccine may comprise (at least) one bi- or evenmulticistronic mRNA, preferably mRNA, i.e. (at least) one mRNA whichcarries, e.g. two or even more of the coding sequences of at least two(preferably different) antigens, preferably selected from one of theabove mentioned antigen combinations. Such coding sequences, e.g. of theat least two (preferably different) antigens, of the (at least) one bi-or even multicistronic mRNA may be separated by at least one IRES(internal ribosomal entry site) sequence, as defined below. Thus, theterm “encoding at least two (preferably different) antigens” may mean,without being limited thereto, that the (at least) one (bi- or evenmulticistronic) mRNA, may encode e.g. at least two, three, four, five,six, seven, eight, nine, ten, eleven or twelve or more (preferablydifferent) antigens of the above mentioned group(s) of antigens, ortheir fragments or variants, etc. In this context, a so-called IRES(internal ribosomal entry site) sequence as defined herein can functionas a sole ribosome binding site, but it can also serve to provide a bi-or even multicistronic RNA as defined herein which codes for severalproteins, which are to be translated by the ribosomes independently ofone another. Examples of IRES sequences which can be used according tothe invention are those from picornaviruses (e.g. FMDV), pestiviruses(CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot andmouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swinefever viruses (CSFV), mouse leukemia virus (MLV), simianimmunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV).

According to a further particularly preferred aspect, the at least onemRNA of the inventive vaccine may comprise a mixture of at least onemonocistronic mRNA as defined herein, and at least one bi- or evenmulticistronic RNA, preferably mRNA, as defined herein. The at least onemonocistronic RNA and/or the at least one bi- or even multicistronic RNApreferably encode different antigens, or their fragments or variants,the antigens preferably being selected from one of the above mentionedantigens, more preferably in one of the above mentioned combinations.However, the at least one monocistronic RNA and the at least one bi- oreven multicistronic RNA may preferably also encode (in part) identicalantigens selected from one of the above mentioned antigens, preferablyin one of the above mentioned combinations, provided that the at leastone mRNA of the inventive vaccine as a whole provides at least two(preferably different) antigens, as defined herein. Such an aspect maybe advantageous e.g. for a staggered, e.g. time dependent,administration of one or several of the at least one mRNA of theinventive vaccine to a patient in need thereof. The components of such avaccine may be contained in (different parts of) a kit of partscomposition or may be e.g. administered separately as components of thesame inventive vaccine as defined according to the present invention.

In a further preferred embodiment the at least one mRNA of the inventivevaccine (or any further nucleic acid as defined herein) may also occurin the form of a modified nucleic acid.

According to a first aspect, the at least one mRNA of the inventivevaccine (or any further nucleic acid as defined herein) may be providedas a “stabilized nucleic acid” that is essentially resistant to in vivodegradation (e.g. by an exo- or endo-nuclease).

In this context, the at least one mRNA of the inventive vaccine (or anyfurther nucleic acid as defined herein) may contain backbonemodifications, sugar modifications or base modifications. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained in theat least one mRNA of the inventive vaccine (or any further nucleic acidas defined herein) are chemically modified. A sugar modification inconnection with the present invention is a chemical modification of thesugar of the nucleotides of the at least one mRNA of the inventivevaccine (or any further nucleic acid as defined herein). Furthermore, abase modification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the at least onemRNA of the inventive vaccine (or any further nucleic acid as definedherein).

According to a further aspect, the at least one mRNA of the inventivevaccine (or any further nucleic acid as defined herein) can contain alipid modification. Such a lipid-modified nucleic acid typicallycomprises a nucleic acid as defined herein, e.g. an mRNA or any furthernucleic acid. Such a lipid-modified mRNA of the inventive vaccine (orany further lipid-modified nucleic acid as defined herein) typicallyfurther comprises at least one linker covalently linked with thatnucleic acid molecule, and at least one lipid covalently linked with therespective linker. Alternatively, the lipid-modified mRNA of theinventive vaccine (or any further lipid-modified nucleic acid as definedherein) comprises at least one nucleic acid molecule as defined herein,e.g. an mRNA or any further nucleic acid, and at least one(bifunctional) lipid covalently linked (without a linker) with thatnucleic acid molecule. According to a third alternative, thelipid-modified mRNA of the inventive vaccine (or any furtherlipid-modified nucleic acid as defined herein) comprises a nucleic acidmolecule as defined herein, e.g. an mRNA or any further nucleic acid, atleast one linker covalently linked with that nucleic acid molecule, andat least one lipid covalently linked with the respective linker, andalso at least one (bifunctional) lipid covalently linked (without alinker) with that nucleic acid molecule.

The at least one mRNA of the inventive vaccine (or any further nucleicacid as defined herein) may likewise be stabilized in order to preventdegradation of the mRNA (or any further nucleic acid molecule) byvarious approaches. It is known in the art that instability and (fast)degradation of RNA in general may represent a serious problem in theapplication of RNA based compositions. This instability of RNA istypically due to RNA-degrading enzymes, “RNAases” (ribonucleases),wherein contamination with such ribonucleases may sometimes completelydegrade RNA in solution. Accordingly, the natural degradation of RNA inthe cytoplasm of cells is very finely regulated and RNase contaminationsmay be generally removed by special treatment prior to use of saidcompositions, in particular with diethyl pyrocarbonate (DEPC). A numberof mechanisms of natural degradation are known in this connection in theprior art, which may be utilized as well. E.g., the terminal structureis typically of critical importance particularly for an mRNA. As anexample, at the 5′ end of naturally occurring mRNAs there is usually aso-called “cap structure” (a modified guanosine nucleotide), and at the3′ end is typically a sequence of up to 200 adenosine nucleotides (theso-called poly-A tail).

According to another aspect, the at least one mRNA of the inventivevaccine may be modified and thus stabilized by modifying the G/C contentof the mRNA, preferably of the coding region thereof.

In a particularly preferred aspect of the present invention, the G/Ccontent of the coding region of the at least one mRNA of the inventivevaccine is modified, particularly increased, compared to the G/C contentof the coding region of its particular wild type coding sequence, i.e.the unmodified mRNA. The encoded amino acid sequence of the mRNA ispreferably not modified compared to the coded amino acid sequence of theparticular wild type mRNA.

The modification of the G/C-content of the at least one mRNA of theinventive vaccine is based on the fact that the sequence of any mRNAregion to be translated is important for efficient translation of thatmRNA. Thus, the composition and the sequence of various nucleotides areimportant. In particular, sequences having an increased G (guanosine)/C(cytosine) content are more stable than sequences having an increased A(adenosine)/U (uracil) content. According to the invention, the codonsof the coding sequence or mRNA are therefore varied compared to its wildtype coding sequence or mRNA, while retaining the translated amino acidsequence, such that they include an increased amount of G/C nucleotides.In respect to the fact that several codons code for one and the sameamino acid (so-called degeneration of the genetic code), the mostfavourable codons for the stability can be determined (so-calledalternative codon usage).

Preferably, the G/C content of the coding region of the at least onemRNA of the inventive vaccine is increased by at least 7%, morepreferably by at least 15%, particularly preferably by at least 20%,compared to the G/C content of the coded region of the wild type mRNA.According to a specific aspect at least 5%, 10%, 20%, 30%, 40%, 50%,60%, more preferably at least 70%, even more preferably at least 80% andmost preferably at least 90%, 95% or even 100% of the substitutablecodons in the region coding for a protein or peptide as defined hereinor its fragment or variant thereof or the whole sequence of the wildtype mRNA sequence or coding sequence are substituted, therebyincreasing the G/C content of said sequence.

In this context, it is particularly preferable to increase the G/Ccontent of the at least one mRNA of the inventive vaccine to the maximum(i.e. 100% of the substitutable codons), in particular in the regioncoding for a protein, compared to the wild type sequence.

According to the invention, a further preferred modification of the atleast one mRNA of the inventive vaccine, especially if the nucleic acidis in the form of an mRNA or codes for an mRNA, is based on the findingthat the translation efficiency is also determined by a differentfrequency in the occurrence of tRNAs in cells. Thus, if so-called “rarecodons” are present in the at least one mRNA of the inventive vaccine toan increased extent, the corresponding modified mRNA is translated to asignificantly poorer degree than in the case where codons coding forrelatively “frequent” tRNAs are present.

Preferably, the coding region of the at least one mRNA of the inventivevaccine is modified compared to the corresponding region of the wildtype mRNA or coding sequence such that at least one codon of the wildtype sequence which codes for a tRNA which is relatively rare in thecell is exchanged for a codon which codes for a tRNA which is relativelyfrequent in the cell and carries the same amino acid as the relativelyrare tRNA. By this modification, the sequences of the at least one mRNAof the inventive vaccine, especially if the nucleic acid is in the formof an mRNA or codes for an mRNA, is modified such that codons for whichfrequently occurring tRNAs are available are inserted. In other words,according to the invention, by this modification all codons of the wildtype sequence which code for a tRNA which is relatively rare in the cellcan in each case be exchanged for a codon which codes for a tRNA whichis relatively frequent in the cell and which, in each case, carries thesame amino acid as the relatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe modified at least one mRNA of the inventive vaccine with the“frequent” codons without modifying the amino acid sequence of theprotein encoded by the coding region of the mRNA. This preferred aspectallows provision of a particularly efficiently translated and stabilized(modified) at least one mRNA of the inventive vaccine.

According to a further preferred aspect of the invention, the at leastone mRNA of the inventive vaccine as defined herein or ay furthernucleic acid molecule as defined herein preferably has at least one 5′and/or 3′ stabilizing sequence. These stabilizing sequences in the 5′and/or 3′ untranslated regions have the effect of increasing thehalf-life of the nucleic acid in the cytosol. These stabilizingsequences can have 100% sequence identity to naturally occurringsequences which occur in viruses, bacteria and eukaryotes, but can alsobe partly or completely synthetic. The untranslated sequences (UTR) ofthe (alpha-)globin gene, e.g. from Homo sapiens or Xenopus laevis may bementioned as an example of stabilizing sequences which can be used inthe present invention for a stabilized nucleic acid. Another example ofa stabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 383), which iscontained in the 3′UTR of the very stable RNA which codes for(alpha-)globin, type(I)-collagen, 15-lipoxygenase or for tyrosinehydroxylase (cf. Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94:2410 to 2414). Such stabilizing sequences can of course be usedindividually or in combination with one another and also in combinationwith other stabilizing sequences known to a person skilled in the art.

Nevertheless, substitutions, additions or eliminations of bases arepreferably carried out with the at least one mRNA of the inventivevaccine or any further nucleic acid molecule as defined herein,especially if the nucleic acid is in the form of an mRNA, using a DNAmatrix for preparation of the nucleic acid molecule by techniques of thewell known site directed mutagenesis or with an oligonucleotide ligationstrategy (see e.g. Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold SpringHarbor, N.Y., 2001). In such a process, for preparation of the at leastone mRNA of the inventive vaccine as defined herein a corresponding DNAmolecule may be transcribed in vitro. This DNA matrix preferablycomprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitrotranscription, which is followed by the desired nucleotide sequence forthe at least one mRNA to be prepared and a termination signal for invitro transcription. The DNA molecule, which forms the matrix of the atleast one mRNA of interest, may be prepared by fermentativeproliferation and subsequent isolation as part of a plasmid which can bereplicated in bacteria. Plasmids which may be mentioned as suitable forthe present invention are e.g. the plasmids pT7 Ts (GenBank accessionnumber U26404; Lai et al., Development 1995, 121: 2349 to 2360), PGEM®series, e.g. PGEM®1 (GenBank accession number X65300; from Promega) andpSP64 (GenBank accession number X65327); cf. also Mezei and Storts,Purification of PCR Products, in: Griffin and Griffin (ed.), PCRTechnology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

Nucleic acid molecules used according to the invention and as definedherein, e.g. the at least one mRNA of the inventive vaccine or anyfurther nucleic acid molecule as defined herein, may be modified asoutlined above for the at least one mRNA of the inventive vaccine.

Additionally, nucleic acid molecules used according to the invention andas defined herein, e.g. the at least one mRNA of the inventive vaccineor any further nucleic acid molecule as defined herein, may be preparedusing any method known in the art, including synthetic methods such ase.g. solid phase synthesis, as well as in vitro methods, such as invitro transcription reactions.

According to one preferred aspect of the present invention the at leastone mRNA of the inventive vaccine may be administered naked withoutbeing associated with any further vehicle, transfection or complexationagent for increasing the transfection efficiency of the at least onemRNA.

In a further preferred aspect of the present invention the at least onemRNA of the inventive vaccine is associated with a vehicle, transfectionor complexation agent for increasing the transfection efficiency of theat least one mRNA. Particularly preferred agents in this contextsuitable for increasing the transfection efficiency are cationic orpolycationic compounds, including protamine, nucleoline, spermine orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides,Pestivirus Erns, HSV, VP22 (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs), PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers,Calcitonin peptide(s), Antennapedia-derived peptides (particularly fromDrosophila antennapedia), pAntp, pIsl, FGF, Lactoferrin, Transportan,Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, orhistones. Additionally, preferred cationic or polycationic proteins orpeptides may be selected from the following proteins or peptides havingthe following total formula:(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), wherein1+m+n+o+x=8-15, and 1, m, n or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred cationic peptides in this context are e.g. Arg₇,Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. Furtherpreferred cationic or polycationic compounds, which can be used astransfection agent may include cationic polysaccharides, for examplechitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI),cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(atrimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

The at least one mRNA of the inventive vaccine encoding at least oneantigen may also be complexed with a polymeric carrier formed bydisulfide-crosslinked cationic components. The term “cationic component”typically refers to a charged molecule, which is positively charged(cation) at a pH value of about 1 to 9, preferably of a pH value of orbelow 9, of or below 8, of or below 7, most preferably at physiologicalpH values, e.g. about 7.3 to 7.4. Accordingly, a cationic peptide,protein or polymer according to the present invention is positivelycharged under physiological conditions, particularly under physiologicalsalt conditions of the cell in vivo. The definition “cationic” may alsorefer to “polycationic” components.

In this context the cationic components, which form basis for thepolymeric carrier of the inventive vaccine by disulfide-crosslinkage,are typically selected from any suitable cationic or polycationicpeptide, protein or polymer suitable for this purpose, particular anycationic or polycationic peptide, protein or polymer capable to complexa nucleic acid as defined according to the present invention, andthereby preferably condensing the nucleic acid. The cationic orpolycationic peptide, protein or polymer, is preferably a linearmolecule, however, branched cationic or polycationic peptides, proteinsor polymers may also be used.

Each cationic or polycationic protein, peptide or polymer of thepolymeric carrier, which may be used to complex the at least one mRNA ofthe inventive vaccine contains at least one —SH moiety, most preferablyat least one cysteine residue or any further chemical group exhibitingan —SH moiety, capable to form a disulfide linkage upon condensationwith at least one further cationic or polycationic protein, peptide orpolymer as cationic component of the polymeric carrier as mentionedherein.

Each cationic or polycationic protein, peptide or polymer or any furthercomponent of the polymeric carrier, which may be used to complex the atleast one mRNA of the inventive vaccine is preferably linked to itsneighbouring component(s) (cationic proteins, peptides, polymers orother components) via disulfide-crosslinking. Preferably, thedisulfide-crosslinking is a (reversible) disulfide bond (—S—S—) betweenat least one cationic or polycationic protein, peptide or polymer and atleast one further cationic or polycationic protein, peptide or polymeror other component of the polymeric carrier. The disulfide-crosslinkingis typically formed by condensation of —SH-moieties of the components ofthe polymeric carrier particularly of the cationic components. Such an—SH-moiety may be part of the structure of the cationic or polycationicprotein, peptide or polymer or any further component of the polymericcarrier prior to disulfide-crosslinking or may be added prior todisulfide-crosslinking by a modification as defined below. In thiscontext, the sulphurs adjacent to one component of the polymericcarrier, necessary for providing a disulfide bond, may be provided bythe component itself, e.g. by a —SH moiety as defined herein or may beprovided by modifying the component accordingly to exhibit a —SH moiety.These —SH-moieties are typically provided by each of the components,e.g. via a cysteine or any further (modified) amino acid of thecomponent, which carries a —SH moiety. In the case that the cationiccomponent or any further component of the polymeric carrier is a peptideor protein it is preferred that the —SH moiety is provided by at leastone cysteine residue. Alternatively, the component of the polymericcarrier may be modified accordingly with a —SH moiety, preferably via achemical reaction with a compound carrying a —SH moiety, such that eachof the components of the polymeric carrier carries at least one such —SHmoiety. Such a compound carrying a —SH moiety may be e.g. an(additional) cysteine or any further (modified) amino acid or compoundof the component of the polymeric carrier, which carries a —SH moiety.Such a compound may also be any non-amino compound or moiety, whichcontains or allows to introduce a —SH moiety into the component asdefined herein. Such non-amino compounds may be attached to thecomponent of the polymeric carrier according to the present inventionvia chemical reactions or binding of compounds, e.g. by binding of a3-thio propionic acid or 2-iminothiolane (Traut's reagent), by amideformation (e.g. carboxylic acids, sulphonic acids, amines, etc.), byMichael addition (e.g maleinimide moieties, α,β unsatured carbonyls,etc.), by click chemistry (e.g. azides or alkines), by alkene/alkinemethatesis (e.g. alkenes or alkines), imine or hydrozone formation(aldehydes or ketons, hydrazins, hydroxylamins, amines), complexationreactions (avidin, biotin, protein G) or components which allowS_(n)-type substitution reactions (e.g halogenalkans, thiols, alcohols,amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphoniumsalts) or other chemical moieties which can be utilized in theattachment of further components. In some cases the —SH moiety may bemasked by protecting groups during chemical attachment to the component.Such protecting groups are known in the art and may be removed afterchemical coupling. In each case, the —SH moiety, e.g. of a cysteine orof any further (modified) amino acid or compound, may be present at theterminal ends or internally at any position of the component of thepolymeric carrier. As defined herein, each of the components of thepolymeric carrier typically exhibits at least one —SH-moiety, but mayalso contain two, three, four, five, or even more —SH-moieties.Additionally to binding of cationic components a —SH moiety may be usedto attach further components of the polymeric carrier of the inventivevaccine as defined herein, particularly an amino acid component, e.g.antigen epitopes, antigens, antibodies, cell penetrating peptides (e.g.TAT), ligands, etc.

As defined above, the polymeric carrier, which may be used to complexthe at least one mRNA of the inventive vaccine may be formed bydisulfide-crosslinked cationic (or polycationic) components.

According to one first alternative, at least one cationic (orpolycationic) component of the polymeric carrier, which may be used tocomplex the at least one mRNA of the inventive vaccine may be selectedfrom cationic or polycationic peptides or proteins. Such cationic orpolycationic peptides or proteins preferably exhibit a length of about 3to 100 amino acids, preferably a length of about 3 to 50 amino acids,more preferably a length of about 3 to 25 amino acids, e.g. a length ofabout 3 to 10, 5 to 15, 10 to 20 or 15 to 25 amino acids. Alternativelyor additionally, such cationic or polycationic peptides or proteins mayexhibit a molecular weight of about 0.01 kDa to about 100 kDa, includinga molecular weight of about 0.5 kDa to about 100 kDa, preferably ofabout 10 kDa to about 50 kDa, even more preferably of about 10 kDa toabout 30 kDa.

In the specific case that the cationic component of the polymericcarrier, which may be used to complex the at least one mRNA of theinventive vaccine comprises a cationic or polycationic peptide orprotein, the cationic properties of the cationic or polycationic peptideor protein or of the entire polymeric carrier, if the polymeric carrieris entirely composed of cationic or polycationic peptides or proteins,may be determined upon its content of cationic amino acids. Preferably,the content of cationic amino acids in the cationic or polycationicpeptide or protein and/or the polymeric carrier is at least 10%, 20%, or30%, preferably at least 40%, more preferably at least 50%, 60% or 70%,but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99%or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to90%, more preferably in the range of about 15% to 75%, even morepreferably in the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, orin a range formed by any two of the afore mentioned values, provided,that the content of all amino acids, e.g. cationic, lipophilic,hydrophilic, aromatic and further amino acids, in the cationic orpolycationic peptide or protein, or in the entire polymeric carrier, ifthe polymeric carrier is entirely composed of cationic or polycationicpeptides or proteins, is 100%.

Preferably, such cationic or polycationic peptides or proteins of thepolymeric carrier, which comprise or are additionally modified tocomprise at least one —SH moiety, are selected from, without beingrestricted thereto, cationic peptides or proteins such as protamine,nucleoline, spermine or spermidine, oligo- or poly-L-lysine (PLL), basicpolypeptides, oligo or polyarginine, cell penetrating peptides (CPPs),chimeric CPPs, such as Transportan, or MPG peptides, HIV-bindingpeptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, members of thepenetratin family, e.g. Penetratin, Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIsl, etc.,antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1),pVEC, hCT-derived peptides, SAP, MAP, PpTG20, Loligomere, FGF,Lactoferrin, histones, VP22 derived or analog peptides, Pestivirus Erns,HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains(PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.

Alternatively or additionally, such cationic or polycationic peptides orproteins of the polymeric carrier, which comprise or are additionallymodified to comprise at least one —SH moiety, are selected from, withoutbeing restricted thereto, following cationic peptides having thefollowing sum formula (I):{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};wherein l+m+n+o+x=3-100, and 1, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may bepositioned at any place of the peptide. In this context cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred. Even more preferred peptides of this formula areoligoarginines such as e.g. Arg₇, Arg₈, Arg₉, Arg₁₂, His₃Arg₉, Arg₉His₃,His₃Arg₉His₃, His₆Arg₉His₆, His₃Arg₄His₃, His₆Arg₄His₆,TyrSer₂Arg₉Ser₂Tyr, (ArgLysHis)₄, Tyr(ArgLysHis)₂Arg, etc.

According to a particular preferred embodiment, such cationic orpolycationic peptides or proteins of the polymeric carrier having theempirical sum formula (I) as shown above, may, without being restrictedthereto, comprise at least one of the following subgroup of formulae:

Arg₇,Arg₈,Arg₉,Arg₁₀,Arg₁₁,Arg₁₂,Arg₁₃,Arg₁₄,Arg₁₅₋₃₀;Lys₇,Lys₈,Lys₉,Lys₁₀,Lys₁₁,Lys₁₂,Lys₁₃,Lys₁₄,Lys₁₅₋₃₀;His₇,His₈,His₉,His₁₀,His₁₁,His₁₂,His₁₃,His₁₄,His₁₅₋₃₀;Orn₇,Orn₈,Orn₉,Orn₁₀,Orn₁₁,Orn₁₂,Orn₁₃,Orn₁₄,Orn₁₅₋₃₀;

According to a further particularly preferred embodiment, cationic orpolycationic peptides or proteins of the polymeric carrier, having theempirical sum formula (I) as shown above and which comprise or areadditionally modified to comprise at least one —SH moiety, may bepreferably selected from, without being restricted thereto, at least oneof the following subgroup of formulae. The following formulae (as withempirical formula (I)) do not specify any amino acid order, but areintended to reflect empirical formulae by exclusively specifying the(number of) amino acids as components of the respective peptide.Accordingly, as an example, empirical formula Arg₍₇₂₉₎Lys₁ is intendedto mean that peptides falling under this formula contain 7 to 19 Argresidues and 1 Lys residue of whatsoever order. If the peptides contain7 Arg residues and 1 Lys residue, all variants having 7 Arg residues and1 Lys residue are encompassed. The Lys residue may therefore bepositioned anywhere in the e.g. 8 amino acid long sequence composed of 7Arg and 1 Lys residues. The subgroup preferably comprises:

Arg₍₄₋₂₉₎Lys₁,Arg₍₄₋₂₉₎His₁,Arg₍₄₋₂₉₎Orn₁,Lys₍₄₋₂₉₎His₁,Lys₍₄₋₂₉₎Orn₁,His₍₄₋₂₉₎Orn₁,Arg₍₃₋₂₈₎Lys₂,Arg₍₃₋₂₈₎His₂,Arg₍₃₋₂₈₎Orn₂,Lys₍₃₋₂₈₎His₂,Lys₍₃₋₂₈₎Orn₂,His₍₃₋₂₈₎Orn₂,Arg₍₂₋₂₇₎Lys₃,Arg₍₂₋₂₇₎His₃,Arg₍₂₋₂₇₎Orn₃,Lys₍₂₋₂₇₎His₃,Lys₍₂₋₂₇₎Orn₃,His₍₂₋₂₇₎Orn₃,Arg₍₁₋₂₆₎Lys₄,Arg₍₁₋₂₆₎His₄,Arg₍₁₋₂₆₎Orn₄,Lys₍₁₋₂₆₎His₄,Lys₍₁₋₂₆₎Orn₄,His₍₁₋₂₆₎Orn₄,

Arg₍₃₋₂₈₎Lys₁His₁,Arg₍₃₋₂₈₎Lys₁Orn₁,Arg₍₃₋₂₈₎His₁Orn₁,Arg₁Lys₍₃₋₂₈₎His₁,Arg₁Lys₍₃₋₂₈₎Orn₁,Lys₍₃₋₂₈₎His₁Orn₁,Arg₁Lys₁His₍₃₋₂₈₎,Arg₁His₍₃₋₂₈₎Orn₁,Lys₁His₍₃₋₂₈₎Orn₁;

Arg₍₂₋₂₇₎Lys₂His₁,Arg₍₂₋₂₇₎Lys₁His₂,Arg₍₂₋₂₇₎Lys₂Orn₁,Arg₍₂₋₂₇₎Lys₁Orn₂,Arg₍₂₋₂₇₎His₂Orn₁,Arg₍₂₋₂₇₎His₁Orn₂,Arg₂Lys₍₂₋₂₇₎His₁,Arg₁Lys₍₂₋₂₇₎His₂,Arg₂Lys₍₂₋₂₇₎Orn₁,Arg₁Lys₍₂₋₂₇₎Orn₂,Lys₍₂₋₂₇₎His₂Orn₁,Lys₍₂₋₂₇₎His₁Orn₂,Arg₂Lys₁His₍₂₋₂₇₎,Arg₁Lys₂His₍₂₋₂₇₎,Arg₂His₍₂₋₂₇₎Orn₁,Arg₁His₍₂₋₂₇₎Orn₂,Lys₂His₍₂₋₂₇₎Orn₁,Lys₁His₍₂₋₂₇₎Orn₂;

Arg₍₁₋₂₆₎Lys₃His₁,Arg₍₁₋₂₆₎Lys₂His₂,Arg₍₁₋₂₆₎Lys₁His₃,Arg₍₁₋₂₆₎Lys₃Orn₁,Arg₍₁₋₂₆₎Lys₂Orn₂,Arg₍₁₋₂₆₎Lys₁Orn₃,Arg₍₁₋₂₆₎His₃Orn₁,Arg₍₁₋₂₆₎His₂Orn₂,Arg₍₁₋₂₆₎His₁Orn₃,Arg₃Lys₍₁₋₂₆₎His₁,Arg₂Lys₍₁₋₂₆₎His₂,Arg₁Lys₍₁₋₂₆₎His₃,Arg₃Lys₍₁₋₂₆₎Orn₁,Arg₂Lys₍₁₋₂₆₎Orn₂,Arg₁Lys₍₁₋₂₆₎Orn₃,Lys₍₁₋₂₆₎His₃Orn₁,Lys₍₁₋₂₆₎His₂Orn₂,Lys₍₁₋₂₆₎His₁Orn₃,Arg₃Lys₁His₍₁₋₂₆₎,Arg₂Lys₂His₍₁₋₂₆₎,Arg₁Lys₃His₍₁₋₂₆₎,Arg₃His₍₁₋₂₆₎Orn₁,Arg₂His₍₁₋₂₆₎Orn₂,Arg₁His₍₁₋₂₆₎Orn₃,Lys₃His₍₁₋₂₆₎Orn₁,Lys₂His₍₁₋₂₆₎Orn₂,Lys₁His₍₁₋₂₆₎Orn₃;

Arg₍₂₋₂₇₎Lys₁His₁Orn₁,Arg₁Lys₍₂₋₂₇₎His₁Orn₁,Arg₁Lys₁His₍₂₋₂₇₎Orn₁,Arg₁Lys₁His₁Orn₍₂₋₂₇₎;

Arg₍₁₋₂₆₎Lys₂His₁Orn₁,Arg₍₁₋₂₆₎Lys₁His₂Orn₁,Arg₍₁₋₂₆₎Lys₁His₁Orn₂,Arg₂Lys₍₁₋₂₆₎His₁Orn₁,Arg₁Lys₍₁₋₂₆₎His₂Orn₁,Arg₁Lys₍₁₋₂₆₎His₁Orn₂,Arg₂Lys₁His₍₁₋₂₆₎Orn₁,Arg₁Lys₂His₍₁₋₂₆₎Orn₁,Arg₁Lys₁His₍₁₋₂₆₎Orn₂,Arg₂Lys₁His₁Orn₍₁₋₂₆₎,Arg₁Lys₂His₁Orn₍₁₋₂₆₎,Arg₁Lys₁His₂Orn₍₁₋₂₆₎;

According to a further particular preferred embodiment, cationic orpolycationic peptides or proteins of the polymeric carrier, having theempirical sum formula (I) as shown above and which comprise or areadditionally modified to comprise at least one —SH moiety, may be,without being restricted thereto, selected from the subgroup consistingof generic formulas Arg₇ (also termed as R₇), Arg₉ (also termed R₉),Arg₁₂ (also termed as R₁₂).

According to a one further particular preferred embodiment, the cationicor polycationic peptide or protein of the polymeric carrier, whendefined according to formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (I)) asshown above and which comprise or are additionally modified to compriseat least one —SH moiety, may be, without being restricted thereto,selected from subformula (Ia):{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)}  formula(Ia)wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80 and 81-90, provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide.

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (I)) as shownabove, comprises or has been modified with at least one cysteine as —SHmoiety in the above meaning such that the cationic or polycationicpeptide as cationic component carries at least one cysteine, which iscapable to form a disulfide bond with other components of the polymericcarrier.

According to another particular preferred embodiment, the cationic orpolycationic peptide or protein of the polymeric carrier, when definedaccording to formula {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}(formula (I)) as shown above, may be, without being restricted thereto,selected from subformula (Ib):Cys¹{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys²  (formula(Ib))wherein empirical formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (I)) is asdefined herein and forms a core of an amino acid sequence according to(semiempirical) formula (I) and wherein Cys¹ and Cys² are Cysteinesproximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x). Exemplary examplesmay comprise any of the above sequences flanked by two Cys and followingsequences:

CysArg₇Cys (SEQ ID NO. 1) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₈Cys(SEQ ID NO. 2) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₉Cys:(SEQ ID NO. 3) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₀Cys(SEQ ID NO. 4) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-CysCysArg₁₁Cys (SEQ ID NO. 5)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Cys CysArg₁₂Cys:(SEQ ID NO. 6) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-CysCysArg₁₃Cys: (SEQ ID NO: 7)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-CysCysArg₁₄Cys: (SEQ ID NO: 8)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-CysCysArg₁₅Cys: (SEQ ID NO. 9)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Arg-CysCysArg₁₆Cys: (SEQ ID NO. 10)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Arg-Arg-CysCysArg₁₇Cys: (SEQ ID NO. 11)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₈Cys: (SEQ ID NO. 12)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₉Cys: (SEQ ID NO. 13)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₂₀Cys: (SEQ ID NO. 14)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg Cys 

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, which may beused to complex the at least one mRNA of the inventive vaccine, e.g.when defined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (I)) as shownabove, has been modified with at least two cysteines as —SH moieties inthe above meaning such that the cationic or polycationic peptide of theinventive polymeric carrier carries at least two (terminal) cysteines,which are capable to form a disulfide bond with other components of thepolymeric carrier.

According to a second alternative, at least one cationic (orpolycationic) component of the polymeric carrier may be selected frome.g. any (non-peptidic) cationic or polycationic polymer suitable inthis context, provided that this (non-peptidic) cationic or polycationicpolymer exhibits or is modified to exhibit at least one —SH-moiety,which provide for a disulfide bond linking the cationic or polycationicpolymer with another component of the polymeric carrier as definedherein. Thus, likewise as defined herein, the polymeric carrier maycomprise the same or different cationic or polycationic polymers.

In the specific case that the cationic component of the polymericcarrier comprises a (non-peptidic) cationic or polycationic polymer thecationic properties of the (non-peptidic) cationic or polycationicpolymer may be determined upon its content of cationic charges whencompared to the overall charges of the components of the cationicpolymer. Preferably, the content of cationic charges in the cationicpolymer at a (physiological) pH as defined herein is at least 10%, 20%,or 30%, preferably at least 40%, more preferably at least 50%, 60% or70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%,99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to90%, more preferably in the range of about 30% to 100%, even preferablyin the range of about 50% to 100%, e.g. 50, 60, 70, 80%, 90% or 100%, orin a range formed by any two of the afore mentioned values, provided,that the content of all charges, e.g. positive and negative charges at a(physiological) pH as defined herein, in the entire cationic polymer is100%.

Preferably, the (non-peptidic) cationic component of the polymericcarrier represents a cationic or polycationic polymer, typicallyexhibiting a molecular weight of about 0.1 or 0.5 kDa to about 100 kDa,preferably of about 1 kDa to about 75 kDa, more preferably of about 5kDa to about 50 kDa, even more preferably of about 5 kDa to about 30kDa, or a molecular weight of about 10 kDa to about 50 kDa, even morepreferably of about 10 kDa to about 30 kDa. Additionally, the(non-peptidic) cationic or polycationic polymer typically exhibits atleast one —SH-moiety, which is capable to form a disulfide linkage uponcondensation with either other cationic components or other componentsof the polymeric carrier as defined herein.

In the above context, the (non-peptidic) cationic component of thepolymeric carrier, which may be used to complex the at least one mRNA ofthe inventive vaccine may be selected from acrylates, modifiedacrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)),chitosanes, aziridines or 2-ethyl-2-oxazoline (forming oligoethylenimines or modifed oligoethylenimines), polymers obtained byreaction of bisacrylates with amines forming oligo beta aminoesters orpoly amido amines, or other polymers like polyesters, polycarbonates,etc. Each molecule of these (non-peptidic) cationic or polycationicpolymers typically exhibits at least one —SH-moiety, wherein these atleast one —SH-moiety may be introduced into the (non-peptidic) cationicor polycationic polymer by chemical modifications, e.g. usingimonothiolan, 3-thio propionic acid or introduction of —SH-moietiescontaining amino acids, such as cysteine or any further (modified) aminoacid. Such —SH-moieties are preferably as already defined above.

In the context of the polymeric carrier, the cationic components, whichform basis for the polymeric carrier, which may be used to complex theat least one mRNA of the inventive vaccine by disulfide-crosslinkage,may be the same or different from each other. It is also particularlypreferred that the polymeric carrier of the present invention comprisesmixtures of cationic peptides, proteins or polymers and optionallyfurther components as defined herein, which are crosslinked by disulfidebonds as described herein.

In this context, the inventive polymeric carrier, which may be used tocomplex the at least one mRNA of the inventive vaccine allows to combinedesired properties of different (short) cationic or polycationicpeptides, proteins or polymers or other components. The polymericcarrier, e.g., allows to efficiently compact nucleic acids for thepurpose of efficient transfection of nucleic acids, for adjuvanttherapy, for the purposes of gene therapy, for gene knock-down or othersstrategies without loss of activity, particularly exhibiting anefficient transfection of a nucleic acid into different cell lines invitro but particularly transfection in vivo. The polymeric carrier isfurthermore not toxic to cells, provides for efficient release of itsnucleic acid cargo, is stable during lyophilization and is applicable asimmunostimulating agent or adjuvant. In this context the components ofthe inventive polymeric carrier can be varied in such a way that thecytokine pattern induced can be determined.

In particular, the polymeric carrier formed by disulfide-linked cationiccomponents allows considerably to vary its peptide or polymeric contentand thus to modulate its biophysical/biochemical properties,particularly the cationic properties of the polymeric carrier, quiteeasily and fast, e.g. by incorporating as cationic components the sameor different cationic peptide(s) or polymer(s) and optionally addingother components into the polymeric carrier. Even though consisting ofquite small non-toxic monomer units the polymeric carrier forms a longcationic binding sequence providing a strong condensation of the mRNA asits nucleic acid cargo and complex stability. Under the reducingconditions of the cytosole (e.g. cytosolic GSH), the complex is rapidlydegraded into its (cationic) components, which are further degraded(e.g. oligopeptides). This supports deliberation of the nucleic acidcargo in the cytosol. Due to degradation into small oligopeptides orpolymers in the cytosol, no toxicity is observed as known forhigh-molecular oligopeptides or polymers, e.g. from high-molecularpolyarginine.

Accordingly, the polymeric carrier, which may be used to complex the atleast one mRNA of the inventive vaccine may comprise different (short)cationic or polycationic peptides, proteins or polymers selected fromcationic or polycationic peptides, proteins or (non-peptidic) polymersas defined above, optionally together with further components as definedherein.

Additionally, the polymeric carrier, which may be used to complex the atleast one mRNA of the inventive vaccine as defined above, morepreferably at least one of the different (short) cationic orpolycationic peptides or (non-peptidic) polymers forming basis for thepolymeric carrier via disulfide-crosslinking, may be, preferably priorto the disulfide-crosslinking, modified with at least one furthercomponent. Alternatively, the polymeric carrier as such may be modifiedwith at least one further component. It may also optionally comprise atleast one further component, which typically forms the polymeric carrierdisulfide together with the other the (short) cationic or polycationicpeptides as defined above via disulfide crosslinking.

To allow modification of a cationic or polycationic peptide or a(non-peptidic) polymer as defined above, each of the components of thepolymeric carrier may (preferably already prior todisulfide-crosslinking) also contain at least one further functionalmoiety, which allows attaching such further components as definedherein. Such functional moieties may be selected from functionalitieswhich allow the attachment of further components, e.g. functionalitiesas defined herein, e.g. by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc.), by Michael addition (e.g maleinimidemoieties, α,β unsatured carbonyls, etc.), by click chemistry (e.g.azides or alkines), by alkene/alkine methatesis (e.g. alkenes oralkines), imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(e)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components.

According to a particularly preferred embodiment, the further component,which may be contained in the polymeric carrier, and which may be usedto complex the at least one mRNA of the inventive vaccine or which maybe used to modify the different (short) cationic or polycationicpeptides or (non-peptidic) polymers forming basis for the polymericcarrier or the biophysical/biochemical properties of the polymericcarrier as defined herein, is an amino acid component (AA). According tothe present invention, the amino acid component (AA) comprises a numberof amino acids preferably in a range of about 1 to 100, preferably in arange of about 1 to 50, more preferably selected from a numbercomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-20, ormay be selected from a range formed by any two of the afore mentionedvalues. In this context the amino acids of amino acid component (AA) canbe chosen independently from each other. For example if in the polymericcarrier two or more (AA) components are present they can be the same orcan be different from each other.

The amino acid component (AA) may contain or may be flanked (e.g.terminally) by a —SH containing moiety, which allows introducing thiscomponent (AA) via a disulfide bond into the polymeric carrier asdefined herein. In the specific case that the —SH containing moietyrepresents a cysteine, the amino acid component (AA) may also be read as-Cys-(AA)-Cys- wherein Cys represents Cysteine and provides for thenecessary —SH-moiety for a disulfide bond. The —SH containing moiety maybe also introduced into amino acid component (AA) using any ofmodifications or reactions as shown above for the cationic component orany of its components.

Furthermore, the amino acid component (AA) may be provided with two—SH-moieties (or even more), e.g. in a form represented by formulaHS-(AA)-SH to allow binding to two functionalities via disulfide bonds,e.g. if the amino acid component (AA) is used as a linker between twofurther components (e.g. as a linker between two cationic polymers). Inthis case, one —SH moiety is preferably protected in a first step usinga protecting group as known in the art, leading to an amino acidcomponent (AA) of formula HS-(AA)-S-protecting group. Then, the aminoacid component (AA) may be bound to a further component of the polymericcarrier, to form a first disulfide bond via the non-protected —SHmoiety. The protected-SH-moiety is then typically deprotected and boundto a further free —SH-moiety of a further component of the polymericcarrier to form a second disulfide bond.

Alternatively, the amino acid component (AA) may be provided with otherfunctionalities as already described above for the other components ofthe polymeric carrier, which allow binding of the amino acid component(AA) to any of components of the polymeric carrier.

Thus, according to the present invention, the amino acid component (AA)may be bound to further components of the polymeric carrier, which maybe used to complex the at least one mRNA of the inventive vaccine withor without using a disulfide linkage. Binding without using a disulfidelinkage may be accomplished by any of the reactions described above,preferably by binding the amino acid component (AA) to the othercomponent of the polymeric carrier using an amid-chemistry as definedherein. If desired or necessary, the other terminus of the amino acidcomponent (AA), e.g. the N- or C-terminus, may be used to couple anothercomponent, e.g. a ligand L. For this purpose, the other terminus of theamino acid component (AA) preferably comprises or is modified tocomprise a further functionality, e.g. an alkyn-species (see above),which may be used to add the other component via e.g. click-chemistry.If the ligand is bound via an acid-labile bond, the bond is preferablycleaved off in the endosome and the polymeric carrier presents aminoacid component (AA) at its surface.

The amino acid component (AA) may occur as a further component of thepolymeric carrier, which may be used to complex the at least one mRNA ofthe inventive vaccine as defined above, e.g. as a linker betweencationic components e.g. as a linker between one cationic peptide and afurther cationic peptide, as a linker between one cationic polymer and afurther cationic polymer, as a linker between one cationic peptide and acationic polymer, all preferably as defined herein, or as an additionalcomponent of the polymeric carrier, e.g. by binding the amino acidcomponent (AA) to the polymeric carrier or a component thereof, e.g. viaside chains, SH-moieties or via further moieties as defined herein,wherein the amino acid component (AA) is preferably accordinglymodified.

According to a further and particularly preferred alternative, the aminoacid component (AA), may be used to modify the polymeric carrier,particularly the content of cationic components in the polymeric carrieras defined above.

In this context it is preferable, that the content of cationiccomponents in the polymeric carrier is at least 10%, 20%, or 30%,preferably at least 40%, more preferably at least 50%, 60% or 70%, butalso preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%, or may be in the range of about 30% to 100%,more preferably in the range of about 50% to 100%, even preferably inthe range of about 70% to 100%, e.g. 70, 80, 90 or 100%, or in a rangeformed by any two of the afore mentioned values, provided, that thecontent of all components in the polymeric carrier is 100%.

In the context of the present invention, the amino acid component (AA)may be selected from the following alternatives.

According to a first alternative, the amino acid component (AA) may bean aromatic amino acid component (AA). The incorporation of aromaticamino acids or sequences as amino aromatic acid component (AA) into thepolymeric carrier of the present invention enables a different (second)binding of the polymeric carrier to the nucleic acid due to interactionsof the aromatic amino acids with the bases of the nucleic acid cargo incontrast to the binding thereof by cationic charged sequences of thepolymeric carrier molecule to the phosphate backbone. This interactionmay occur e.g. by intercalations or by minor or major groove binding.This kind of interaction is not prone to decompaction by anioniccomplexing partners (e.g. Heparin, Hyaluronic acids) which are foundmainly in the extracellular matrix in vivo and is also less susceptibleto salt effects.

For this purpose, the amino acids in the aromatic amino acid component(AA) may be selected from either the same or different aromatic aminoacids e.g. selected from Trp, Tyr, or Phe. Alternatively, the aminoacids (or the entire aromatic amino acid component (AA)) may be selectedfrom following peptide combinations Trp-Tyr, Tyr-Trp, Trp-Trp, Tyr-Tyr,Trp-Tyr-Trp, Tyr-Trp-Tyr, Trp-Trp-Trp, Tyr-Tyr-Tyr, Trp-Tyr-Trp-Tyr,Tyr-Trp-Tyr-Trp, Trp-Trp-Trp-Trp, Phe-Tyr, Tyr-Phe, Phe-Phe,Phe-Tyr-Phe, Tyr-Phe-Tyr, Phe-Phe-Phe, Phe-Tyr-Phe-Tyr, Tyr-Phe-Tyr-Phe,Phe-Phe-Phe-Phe, Phe-Trp, Trp-Phe, Phe-Phe, Phe-Trp-Phe, Trp-Phe-Trp,Phe-Trp-Phe-Trp, Trp-Phe-Trp-Phe, or Tyr-Tyr-Tyr-Tyr, etc. (SEQ ID NOs:15-42). Such peptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 or even more times. These peptidecombinations may also be combined with each other as suitable.

Additionally, the aromatic amino acid component (AA) may contain or maybe flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of the polymericcarrier as defined above, e.g. as a linker. Such a —SH containing moietymay be any moiety as defined herein suitable to couple one component asdefined herein to a further component as defined herein. As an example,such a —SH containing moiety may be a cysteine. Then, e.g. the aromaticamino acid component (AA) may be selected from e.g. peptide combinationsCys-Tyr-Cys, Cys-Trp-Cys, Cys-Trp-Tyr-Cys, Cys-Tyr-Trp-Cys,Cys-Trp-Trp-Cys, Cys-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Cys,Cys-Tyr-Trp-Tyr-Cys, Cys-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Cys,Cys-Trp-Tyr-Trp-Tyr-Cys, Cys-Tyr-Trp-Tyr-Trp-Cys,Cys-Trp-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Tyr-Cys, Cys-Phe-Cys,Cys-Phe-Tyr-Cys, Cys-Tyr-Phe-Cys, Cys-Phe-Phe-Cys, Cys-Tyr-Tyr-Cys,Cys-Phe-Tyr-Phe-Cys, Cys-Tyr-Phe-Tyr-Cys, Cys-Phe-Phe-Phe-Cys,Cys-Tyr-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Tyr-Cys, Cys-Tyr-Phe-Tyr-Phe-Cys,or Cys-Phe-Phe-Phe-Phe-Cys, Cys-Phe-Trp-Cys, Cys-Trp-Phe-Cys,Cys-Phe-Phe-Cys, Cys-Phe-Trp-Phe-Cys, Cys-Trp-Phe-Trp-Cys,Cys-Phe-Trp-Phe-Trp-Cys, Cys-Trp-Phe-Trp-Phe-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein. (SEQ ID NOs: 43-75). Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or even more times. These peptide combinations mayalso be combined with each other as suitable.

Additionally, the aromatic amino acid component (AA) may contain orrepresent at least one proline, which may serve as a structure breakerof longer sequences of Trp, Tyr and Phe in the aromatic amino acidcomponent (AA), preferably two, three or more prolines.

According to a second alternative, the amino acid component (AA) may bea hydrophilic (and preferably non charged polar) amino acid component(AA). The incorporation of hydrophilic (and preferably non chargedpolar) amino acids or sequences as amino hydrophilic (and preferably noncharged polar) acid component (AA) into the polymeric carrier of thepresent invention enables a more flexible binding to the nucleic acidcargo. This leads to a more effective compaction of the nucleic acidcargo and hence to a better protection against nucleases and unwanteddecompaction. It also allows provision of a (long) polymeric carrierwhich exhibits a reduced cationic charge over the entire carrier and inthis context to better adjusted binding properties, if desired ornecessary.

For this purpose, the amino acids in the hydrophilic (and preferably noncharged polar) amino acid component (AA) may be selected from either thesame or different hydrophilic (and preferably non charged polar) aminoacids e.g. selected from Thr, Ser, Asn or Gln. Alternatively, the aminoacids (or the entire hydrophilic (and preferably non charged polar)amino acid component (AA)) may be selected from following peptidecombinations Ser-Thr, Thr-Ser, Ser-Ser, Thr-Thr, Ser-Thr-Ser,Thr-Ser-Thr, Ser-Ser-Ser, Thr-Thr-Thr, Ser-Thr-Ser-Thr, Thr-Ser-Thr-Ser,Ser-Ser-Ser-Ser, Thr-Thr-Thr-Thr, Gln-Asn, Asn-Gln, Gln-Gln, Asn-Asn,Gln-Asn-Gln, Asn-Gln-Asn, Gln-Gln-Gln, Asn-Asn-Asn, Gln-Asn-Gln-Asn,Asn-Gln-Asn-Gln, Gln-Gln-Gln-Gln, Asn-Asn-Asn-Asn, Ser-Asn, Asn-Ser,Ser-Ser, Asn-Asn, Ser-Asn-Ser, Asn-Ser-Asn, Ser-Ser-Ser, Asn-Asn-Asn,Ser-Asn-Ser-Asn, Asn-Ser-Asn-Ser, Ser-Ser-Ser-Ser, or Asn-Asn-Asn-Asn,etc. (SEQ ID NOs: 76-111) Such peptide combinations may be repeated e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or even more times.These peptide combinations may also be combined with each other assuitable.

Additionally, the hydrophilic (and preferably non-charged polar) aminoacid component (AA) may contain or may be flanked by a —SH containingmoiety, which allows introducing this component via a disulfide bond asa further part of the polymeric carrier as defined above, e.g. as alinker. Such a —SH containing moiety may be any moiety as defined hereinsuitable to couple one component as defined herein to a furthercomponent as defined herein. As an example, such a —SH containing moietymay be a cysteine. Then, e.g. the hydrophilic (and preferablynon-charged polar) amino acid component (AA) may be selected from e.g.peptide combinations Cys-Thr-Cys, Cys-Ser-Cys, Cys-Ser-Thr-Cys,Cys-Thr-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Thr-Thr-Cys, Cys-Ser-Thr-Ser-Cys,Cys-Thr-Ser-Thr-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Cys,Cys-Ser-Thr-Ser-Thr-Cys, Cys-Thr-Ser-Thr-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Thr-Cys, Cys-Asn-Cys,Cys-Gln-Cys, Cys-Gln-Asn-Cys, Cys-Asn-Gln-Cys, Cys-Gln-Gln-Cys,Cys-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Cys, Cys-Asn-Gln-Asn-Cys,Cys-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Asn-Cys,Cys-Asn-Gln-Asn-Gln-Cys, Cys-Gln-Gln-Gln-Gln-Cys,Cys-Asn-Asn-Asn-Asn-Cys, Cys-Asn-Cys, Cys-Ser-Cys, Cys-Ser-Asn-Cys,Cys-Asn-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Cys,Cys-Asn-Ser-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Cys,Cys-Ser-Asn-Ser-Asn-Cys, Cys-Asn-Ser-Asn-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, or Cys-Asn-Asn-Asn-Asn-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein. (SEQ ID NOs: 112-153) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the hydrophilic (and preferably non-charged polar) aminoacid component (AA) may contain at least one proline, which may serve asa structure breaker of longer sequences of Ser, Thr and Asn in thehydrophilic (and preferably non charged polar) amino acid component(AA), preferably two, three or more prolines.

According to a third alternative, the amino acid component (AA) may be alipophilic amino acid component (AA). The incorporation of lipophilicamino acids or sequences as amino lipophilic acid component (AA) intothe polymeric carrier of the present invention enables a strongercompaction of the nucleic acid cargo and/or the polymeric carrier andits nucleic acid cargo when forming a complex. This is particularly dueto interactions of one or more polymer strands of the polymeric carrier,particularly of lipophilic sections of lipophilic amino acid component(AA) and the nucleic acid cargo. This interaction will preferably add anadditional stability to the complex between the polymeric carrier andits nucleic acid cargo. This stabilization may somehow be compared to asort of non covalent crosslinking between different polymer strands.Especially in aqueous environment this interaction is typically strongand provides a significant effect.

For this purpose, the amino acids in the lipophilic amino acid component(AA) may be selected from either the same or different lipophilic aminoacids e.g. selected from Leu, Val, Ile, Ala, Met. Alternatively, theamino acid AA (or the entire lipophilic amino acid component (AA)) maybe selected from following peptide combinations Leu-Val, Val-Leu,Leu-Leu, Val-Val, Leu-Val-Leu, Val-Leu-Val, Leu-Leu-Leu, Val-Val-Val,Leu-Val-Leu-Val, Val-Leu-Val-Leu, Leu-Leu-Leu-Leu, Val-Val-Val-Val,Ile-Ala, Ala-Ile, Ile-Ile, Ala-Ala, Ile-Ala-Ile, Ala-Ile-Ala,Ile-Ile-Ile, Ala-Ala-Ala, Ile-Ala-Ile-Ala, Ala-Ile-Ala-Ile,Ile-Ile-Ile-Ile, Ala-Ala-Ala-Ala, Met-Ala, Ala-Met, Met-Met, Ala-Ala,Met-Ala-Met, Ala-Met-Ala, Met-Met-Met, Ala-Ala-Ala, Met-Ala-Met-Ala,Ala-Met-Ala-Met, or Met-Met-Met-Met etc. (SEQ ID NOs: 154-188) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the lipophilic amino acid component (AA) may contain ormay be flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of the polymericcarrier above, e.g. as a linker. Such a —SH containing moiety may be anymoiety as defined herein suitable to couple one component as definedherein to a further component as defined herein. As an example, such a—SH containing moiety may be a cysteine. Then, e.g. the lipophilic aminoacid component (AA) may be selected from e.g. peptide combinationsCys-Val-Cys, Cys-Leu-Cys, Cys-Leu-Val-Cys, Cys-Val-Leu-Cys,Cys-Leu-Leu-Cys, Cys-Val-Val-Cys, Cys-Leu-Val-Leu-Cys,Cys-Val-Leu-Val-Cys, Cys-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Cys,Cys-Leu-Val-Leu-Val-Cys, Cys-Val-Leu-Val-Leu-Cys,Cys-Leu-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Val-Cys, Cys-Ala-Cys,Cys-Ile-Cys, Cys-Ile-Ala-Cys, Cys-Ala-Ile-Cys, Cys-Ile-Ile-Cys,Cys-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Cys, Cys-Ala-Ile-Ala-Cys,Cys-Ile-Ile-Ile-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Ala-Cys,Cys-Ala-Ile-Ala-Ile-Cys, Cys-Ile-Ile-Ile-Ile-Cys, orCys-Ala-Ala-Ala-Ala-Cys, Cys-Met-Cys, Cys-Met-Ala-Cys, Cys-Ala-Met-Cys,Cys-Met-Met-Cys, Cys-Ala-Ala-Cys, Cys-Met-Ala-Met-Cys,Cys-Ala-Met-Ala-Cys, Cys-Met-Met-Met-Cys, Cys-Ala-Ala-Ala-Cys,Cys-Met-Ala-Met-Ala-Cys, Cys-Ala-Met-Ala-Met-Cys,Cys-Met-Met-Met-Met-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein. (SEQ ID NOs: 189-229) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the lipophilic amino acid component (AA) may contain atleast one proline, which may serve as a structure breaker of longersequences of Leu, Val, Ile, Ala and Met in the lipophilic amino acidcomponent (AA), preferably two, three or more prolines.

Finally, according to a fourth alternative, the amino acid component(AA) may be a weak basic amino acid component (AA). The incorporation ofweak basic amino acids or sequences as weak basic amino acid component(AA) into the polymeric carrier of the present invention may serve as aproton sponge and facilitates endosomal escape (also called endosomalrelease) (proton sponge effect). Incorporation of such a weak basicamino acid component (AA) preferably enhances transfection efficiency.

For this purpose, the amino acids in the weak basic amino acid component(AA) may be selected from either the same or different weak amino acidse.g. selected from histidine or aspartate (aspartic acid).Alternatively, the weak basic amino acids (or the entire weak basicamino acid component (AA)) may be selected from following peptidecombinations Asp-His, His-Asp, Asp-Asp, His-His, Asp-His-Asp,His-Asp-His, Asp-Asp-Asp, His-His-His, Asp-His-Asp-His, His-Asp-His-Asp,Asp-Asp-Asp-Asp, or His-His-His-His, etc. (SEQ ID NOs: 230-241) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the weak basic amino acid component (AA) may contain ormay be flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of the polymericcarrier as defined above, e.g. as a linker. Such a —SH containing moietymay be any moiety as defined herein suitable to couple one component asdefined herein to a further component as defined herein. As an example,such a —SH containing moiety may be a cysteine. Then, e.g. the weakbasic amino acid component (AA) may be selected from e.g. peptidecombinations Cys-His-Cys, Cys-Asp-Cys, Cys-Asp-His-Cys, Cys-His-Asp-Cys,Cys-Asp-Asp-Cys, Cys-His-His-Cys, Cys-Asp-His-Asp-Cys,Cys-His-Asp-His-Cys, Cys-Asp-Asp-Asp-Cys, Cys-His-His-His-Cys,Cys-Asp-His-Asp-His-Cys, Cys-His-Asp-His-Asp-Cys,Cys-Asp-Asp-Asp-Asp-Cys, or Cys-His-His-His-His-Cys, etc. Each Cys abovemay also be replaced by any modified peptide or chemical compoundcarrying a free —SH-moiety as defined herein. (SEQ ID NOs: 242-255) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the weak basic amino acid component (AA) may contain atleast one proline, which may serve as a structure breaker of longersequences of histidine or aspartate (aspartic acid) in the weak basicamino acid component (AA), preferably two, three or more prolines.

According to a fifth alternative, the amino acid component (AA) may be asignal peptide or signal sequence, a localisation signal or sequence, anuclear localisation signal or sequence (NLS), an antibody, a cellpenetrating peptide, (e.g. TAT), etc. Preferably such an amino acidcomponent (AA) is bound to the polymeric carrier or to another componentof the polymeric carrier via a (reversible) disulfide bond. In thiscontext the signal peptide or signal sequence, a localisation signal orsequence, a nuclear localisation signal or sequence (NLS), an antibody,a cell penetrating peptide, (e.g. TAT), etc.; additionally comprises atleast one —SH-moiety. In this context a signal peptide, a localisationsignal or sequence or a nuclear localisation signal or sequence (NLS),may be used to direct the inventive polymeric carrier cargo complex tospecific target cells (e.g. hepatocytes or antigen-presenting cells) andpreferably allows a translocalisation of the polymeric carrier to aspecific target, e.g. into the cell, into the nucleus, into theendosomal compartment, sequences for the mitochondrial matrix,localisation sequences for the plasma membrane, localisation sequencesfor the Golgi apparatus, the nucleus, the cytoplasm and thecytoskeleton, etc. Such signal peptide, a localisation signal orsequence or a nuclear localisation signal may be used for the transportof any of the herein defined nucleic acids, preferably an RNA or a DNA,more preferably an shRNA or a pDNA, e.g. into the nucleus. Without beinglimited thereto, such a signal peptide, a localisation signal orsequence or a nuclear localisation signal may comprise, e.g.,localisation sequences for the endoplasmic reticulum. Particularlocalisation signals or sequences or a nuclear localisation signals mayinclude e.g. KDEL (SEQ ID NO: 256), DDEL (SEQ ID NO: 257), DEEL (SEQ IDNO: 258), QEDL (SEQ ID NO: 259), RDEL (SEQ ID NO: 260), and GQNLSTSN(SEQ ID NO: 261), nuclear localisation sequences, including PKKKRKV (SEQID NO: 262), PQKKIKS (SEQ ID NO: 263), QPKKP (SEQ ID NO: 264), RKKR (SEQID NO: 265), RKKRRQRRRAHQ (SEQ ID NO: 266), RQARRNRRRRWRERQR (SEQ ID NO:267), MPLTRRRPAASQALAPPTP (SEQ ID NO: 268), GAALTILV (SEQ ID NO: 269),and GAALTLLG (SEQ ID NO: 270), localisation sequences for the endosomalcompartment, including MDDQRDLISNNEQLP (SEQ ID NO: 271), localisationsequences for the mitochondrial matrix, includingMLFNLRXXLNNAAFRHGHNFMVRNFRCGQPLX (SEQ ID NO: 272), localisationsequences for the plasma membrane: GCVCSSNP (SEQ ID NO: 273), GQTVTTPL(SEQ ID NO: 274), GQELSQHE (SEQ ID NO: 275), GNSPSYNP (SEQ ID NO: 276),GVSGSKGQ (SEQ ID NO: 277), GQTITTPL (SEQ ID NO: 278), GQTLTTPL (SEQ IDNO: 279), GQIFSRSA (SEQ ID NO: 280), GQIHGLSP (SEQ ID NO: 281), GARASVLS(SEQ ID NO: 282), and GCTLSAEE (SEQ ID NO: 283), localisation sequencesfor the endoplasmic reticulum and the nucleus, including GAQVSSQK (SEQID NO: 284), and GAQLSRNT (SEQ ID NO: 285), localisation sequences forthe Golgi apparatus, the nucleus, the cytoplasm and the cytoskeleton,including GNAAAAKK (SEQ ID NO: 286), localisation sequences for thecytoplasm and cytoskeleton, including GNEASYPL (SEQ ID NO: 287),localisation sequences for the plasma membrane and cytoskeleton,including GSSKSKPK (SEQ ID NO: 288), etc. Examples of secretory signalpeptide sequences as defined herein include, without being limitedthereto, signal sequences of classical or non-classical MHC-molecules(e.g. signal sequences of MHC I and II molecules, e.g. of the MHC classI molecule HLA-A*0201), signal sequences of cytokines or immunoglobulinsas defined herein, signal sequences of the invariant chain ofimmunoglobulins or antibodies as defined herein, signal sequences ofLamp1, Tapasin, Erp57, Calreticulin, Calnexin, and further membraneassociated proteins or of proteins associated with the endoplasmicreticulum (ER) or the endosomal-lysosomal compartment. Particularlypreferably, signal sequences of MHC class I molecule HLA-A*0201 may beused according to the present invention. Such an additional componentmay be bound e.g. to a cationic polymer or to any other component of thepolymeric carrier as defined herein. Preferably this signal peptide,localisation signal or sequence or nuclear localisation signal orsequence (NLS), is bound to the polymeric carrier or to anothercomponent of the polymeric carrier via a (reversible) disulfide bond.For this purpose the (AA) component additionally comprises at least one—SH moiety as defined herein. The binding to any of components of thepolymeric carrier may also be accomplished using an acid-labile bond,preferably via a side chain of any of components of the polymericcarrier, which allows to detach or release the additional component atlower pH-values, e.g. at physiological pH-values as defined herein.

Additionally, according to another alternative, the amino acid component(AA) may be a functional peptide or protein, which may modulate thefunctionality of the polymeric carrier accordingly. Such functionalpeptides or proteins as the amino acid component (AA) preferablycomprise any peptides or proteins as defined herein, e.g. as definedbelow as therapeutically active proteins. According to one alternative,such further functional peptides or proteins may comprise so called cellpenetrating peptides (CPPs) or cationic peptides for transportation.Particularly preferred are CPPs, which induce a pH-mediatedconformational change in the endosome and lead to an improved release ofthe polymeric carrier (in complex with a nucleic acid) from the endosomeby insertion into the lipid layer of the liposome. These cellpenetrating peptides (CPPs) or cationic peptides for transportation, mayinclude, without being limited thereto protamine, nucleoline, spermineor spermidine, oligo- or poly-L-lysine (PLL), basic polypeptides, oligoor poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, suchas Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat(HIV), Tat-derived peptides, members of the penetratin family, e.g.Penetratin, Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, pIsl, etc., antimicrobial-derived CPPs e.g.Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP,MAP, KALA, PpTG20, Loligomere, FGF, Lactoferrin, histones, VP22 derivedor analog peptides, Pestivirus Erns, HSV, VP22 (Herpes simplex), MAP,KALA or protein transduction domains (PTDs, PpT620, proline-richpeptides, arginine-rich peptides, lysine-rich peptides, Pep-1,L-oligomers, Calcitonin peptide(s), etc. Such an amino acid component(AA) may also be bound to any component of the polymeric carrier asdefined herein. Preferably it is bound to the polymeric carrier or toanother component of the polymeric carrier via a (reversible) disulfidebond. For the above purpose, the amino acid component (AA) preferablycomprises at least one —SH moiety as defined herein. The binding to anyof components of the polymeric carrier may also be accomplished using anSH-moiety or an acid-labile bond, preferably via a side chain of any ofcomponents of the polymeric carrier which allows to detach or releasethe additional component at lower pH-values, e.g. at physiologicalpH-values as defined herein.

According to a last alternative, the amino acid component (AA) mayconsist of any peptide or protein which can execute any favourablefunction in the cell. Particularly preferred are peptides or proteinsselected from therapeutically active proteins or peptides, fromantigens, e.g. tumour antigens, pathogenic antigens (animal antigens,viral antigens, protozoan antigens, bacterial antigens, allergicantigens), autoimmune antigens, or further antigens, from allergens,from antibodies, from immunostimulatory proteins or peptides, fromantigen-specific T cell receptors, or from any other protein or peptidesuitable for a specific (therapeutic) application as defined below forcoding nucleic acids. Particularly preferred are peptide epitopes fromantigens as defined herein.

The polymeric carrier, which may be used to complex the at least onemRNA of the inventive vaccine may comprise at least one of the abovementioned cationic or polycationic peptides, proteins or polymers orfurther components, e.g. (AA), wherein any of the above alternatives maybe combined with each other, and may be formed by polymerizing same in apolymerization condensation reaction via their —SH-moieties.

According to another aspect, the polymeric carrier, which may be used tocomplex the at least one mRNA of the inventive vaccine or singlecomponents thereof, e.g. of the above mentioned cationic or polycationicpeptides, proteins or polymers or further components, e.g. (AA), may befurther modified with a ligand, preferably a carbohydrate, morepreferably a sugar, even more preferably mannose. Preferably this ligandis bound to the polymeric carrier or to a component of the polymericcarrier via a (reversible) disulfide bond or via Michael addition. Inthe case that the ligand is bound by a disulfide bond the ligandadditionally comprises at least one —SH-moiety. These ligands may beused to direct the inventive polymeric carrier cargo complex to specifictarget cells (e.g. hepatocytes or antigen-presenting cells). In thiscontext mannose is particularly preferred as ligand in the case thatdendritic cells are the target especially for vaccination or adjuvantpurposes.

According to one specific aspect, the entire inventive polymeric carriermay be formed by a polymerization condensation (of at least one) of theabove mentioned cationic or polycationic peptides, proteins or polymersor further components, e.g. (AA), via their —SH-moieties in a first stepand complexing the nucleic acid to such a polymeric carrier in a secondstep. The polymeric carrier may thus contain a number of at least one oreven more of the same or different of the above defined cationic orpolycationic peptides, proteins or polymers or further components, e.g.(AA), the number preferably determined by the above range.

According to one alternative specific aspect, the inventive polymericcarrier, which may be used to complex the at least one mRNA of theinventive vaccine is formed by carrying out the polymerizationcondensation of at least one of the above mentioned cationic orpolycationic peptides, proteins or polymers or further components, e.g.(AA), via their —SH-moieties simultaneously to complexing the at leastone mRNA encoding the at least one antigen to the (in situ prepared)polymeric carrier. Likewise, the polymeric carrier may thus also herecontain a number of at least one or even more of the same or differentof the above defined cationic or polycationic peptides, proteins orpolymers or further components, e.g. (AA), the number preferablydetermined by the above range.

According to a further alternative aspect, the inventive polymericcarrier may be selected from a polymeric carrier molecule according togeneric formula (VI):L-P¹—S—[S—P²—S]_(n)—S—P³-Lwherein,

-   P¹ and P³ are different or identical to each other and represent a    linear or branched hydrophilic polymer chain, each P¹ and P³    exhibiting at least one —SH-moiety, capable to form a disulfide    linkage upon condensation with component P², or alternatively with    (AA), (AA)_(x), or [(AA)_(x)], if such components are used as a    linker between P¹ and P² or P³ and P²) and/or with further    components (e.g. (AA), (AA)_(x), [(AA)_(x)], or L), the linear or    branched hydrophilic polymer chain selected independent from each    other from polyethylene glycol (PEG),    poly-N-(2-hydroxypropyl)methacrylamide,    poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl    L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),    hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the    hydrophilic polymer chain exhibits a molecular weight of about 1 kDa    to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or more    preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to about    25 kDa or 5 kDa to about 10 kDa;-   p² is a cationic or polycationic peptide or protein, e.g. as defined    herein, and preferably having a length of about 3 to about 100 amino    acids, more preferably having a length of about 3 to about 50 amino    acids, even more preferably having a length of about 3 to about 25    amino acids, e.g. a length of about 3 to 10, 5 to 15, 10 to 20 or 15    to 25 amino acids, more preferably a length of about 5 to about 20    and even more preferably a length of about 10 to about 20; or    -   is a cationic or polycationic polymer, e.g. as defined herein,        typically having a molecular weight of about 0.5 kDa to about 30        kDa, including a molecular weight of about 1 kDa to about 20        kDa, even more preferably of about 1.5 kDa to about 10 kDa, or        having a molecular weight of about 0.5 kDa to about 100 kDa,        including a molecular weight of about 10 kDa to about 50 kDa,        even more preferably of about 10 kDa to about 30 kDa;    -   each P² exhibiting at least two —SH-moieties, capable to form a        disulfide linkage upon condensation with further components P²        or component(s) P¹ and/or P³ or alternatively with further        components (e.g. (AA), (AA)_(x), or [(AA)_(x)]_(z));-   —S—S— is a (reversible) disulfide bond (the brackets are omitted for    better readability), wherein S preferably represents sulphur or a    —SH carrying moiety, which has formed a (reversible) disulfide bond.    The (reversible) disulfide bond is preferably formed by condensation    of —SH-moieties of either components P¹ and P², P² and P², or P² and    P³, or optionally of further components as defined herein (e.g. L,    (AA), (AA)_(x), [(AA)_(x)]_(z), etc); The —SH-moiety may be part of    the structure of these components or added by a modification as    defined below;-   L is an optional ligand, which may be present or not, and may be    selected independent from the other from RGD, Transferrin, Folate, a    signal peptide or signal sequence, a localization signal or    sequence, a nuclear localization signal or sequence (NLS), an    antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand    of a receptor (e.g. cytokines, hormones, growth factors etc), small    molecules (e.g. carbohydrates like mannose or galactose or synthetic    ligands), small molecule agonists, inhibitors or antagonists of    receptors (e.g. RGD peptidomimetic analogues) etc.;-   n is an integer, typically selected from a range of about 1 to 50,    preferably from a range of about 1, 2 or 3 to 30, more preferably    from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1,    2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or    a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of    about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a    range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of    about 6 to 11 or 7 to 10. Most preferably, n is in a range of about    1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3,    or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of    about 1, 2, or 3 to 7.

As defined above, ligands (L), may be optionally used in the inventivepolymeric carrier molecule according to generic formula (VI), e.g. fordirection of the inventive carrier polymer and its entire “cargo” (theadjuvant component and/or the antigen of the inventive composition orvaccine composition) into specific cells. They may be selectedindependent from the other from RGD, Transferrin, Folate, a signalpeptide or signal sequence, a localization signal or sequence, a nuclearlocalization signal or sequence (NLS), an antibody, a cell penetratingpeptide (CPP), (e.g. TAT, KALA), a ligand of a receptor (e.g. cytokines,hormones, growth factors etc), small molecules (e.g. carbohydrates likemannose or galactose or synthetic ligands), small molecule agonists,inhibitors or antagonists of receptors (e.g. RGD peptidomimeticanalogues) or any such molecule as further defined below, etc.Particularly preferred are cell penetrating peptides (CPPs), whichinduce a pH-mediated conformational change in the endosome and lead toan improved release of the inventive polymeric carrier (in complex witha nucleic acid) from the endosome by insertion into the lipid layer ofthe liposome. Such called CPPs or cationic peptides for transportation,may include, without being limited thereto protamine, nucleoline,spermine or spermidine, poly-L-lysine (PLL), basic polypeptides,poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, such asTransportan, or MPG peptides, HIV-binding peptides, Tat, HIV1 Tat (HIV),Tat-derived peptides, oligoarginines, members of the penetratin family,e.g. Penetratin, Antennapedia-derived peptides (particularly fromDrosophila antennapedia), pAntp, pIsl, etc., antimicrobial-derived CPPse.g. Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides,SAP, MAP, PpTG20, Proline-rich peptides, Loligomers, Arginine-richpeptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine,poly-Arginine, histones, VP22 derived or analog peptides, PestivirusErns, HSV, VP22 (Herpes simplex), MAP, KALA or protein transductiondomains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides,lysine-rich peptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.Particularly preferred in this context is mannose as ligand to targetantigen presenting cells which carries on their cell membrane mannosereceptors. In a further preferred aspect of the first embodiment of thepresent invention galactose as optional ligand can be used to targethepatocytes. Such ligands may be attached to component P¹ and/or P³ byreversible disulfide bonds as defined below or by any other possiblechemical attachment, e.g. by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc), by Michael addition (e.g. maleinimidemoieties, α,β unsatured carbonyls, etc), by click chemistry (e.g. azidesor alkines), by alkene/alkine methatesis (e.g. alkenes or alkines),imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components.

In the context of formula (VI) of the present invention components P¹and P³ represent a linear or branched hydrophilic polymer chain,containing at least one —SH-moiety, each P¹ and P³ independentlyselected from each other, e.g. from polyethylene glycol (PEG),poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethylphosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkylL-glutamine). P¹ and P³ may be identical or different to each other.Preferably, each of hydrophilic polymers P¹ and P³ exhibits a molecularweight of about 1 kDa to about 100 kDa, preferably of about 1 kDa toabout 75 kDa, more preferably of about 5 kDa to about 50 kDa, even morepreferably of about 5 kDa to about 25 kDa. Additionally, each ofhydrophilic polymers P¹ and P³ typically exhibits at least one—SH-moiety, wherein the at least one —SH-moiety is capable to form adisulfide linkage upon reaction with component P² or with component (AA)or (AA)_(x), if used as linker between P¹ and P² or P³ and P² as definedbelow and optionally with a further component, e.g. L and/or (AA) or(AA)_(x), e.g. if two or more —SH-moieties are contained. The followingsubformulae “P¹—S—S—P²” and “P²—S—S—P³” within generic formula (VI)above (the brackets are omitted for better readability), wherein any ofS, P¹ and P³ are as defined herein, typically represent a situation,wherein one —SH-moiety of hydrophilic polymers P¹ and P³ was condensedwith one —SH-moiety of component P² of generic formula (VI) above,wherein both sulphurs of these —SH-moieties form a disulfide bond —S—S—as defined herein in formula (VI). These —SH-moieties are typicallyprovided by each of the hydrophilic polymers P¹ and P³, e.g. via aninternal cysteine or any further (modified) amino acid or compound whichcarries a —SH moiety. Accordingly, the subformulae “P¹—S—S—P²” and“P²—S—S—P³” may also be written as “P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”,if the —SH-moiety is provided by a cysteine, wherein the term Cys-Cysrepresents two cysteines coupled via a disulfide bond, not via a peptidebond. In this case, the term “—S—S—” in these formulae may also bewritten as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, theterm “-Cys-Cys-” does not represent a peptide bond but a linkage of twocysteines via their —SH-moieties to form a disulfide bond. Accordingly,the term “-Cys-Cys-” also may be understood generally as“-(CysS)—(S-Cys)-”, wherein in this specific case S indicates thesulphur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and“—Cys-S” indicate a disulfide bond between a —SH containing moiety and acysteine, which may also be written as “—S—(S-Cys)” and “-(Cys-S)—S”.Alternatively, the hydrophilic polymers P¹ and P³ may be modified with a—SH moiety, preferably via a chemical reaction with a compound carryinga —SH moiety, such that each of the hydrophilic polymers P¹ and P³carries at least one such —SH moiety. Such a compound carrying a —SHmoiety may be e.g. an (additional) cysteine or any further (modified)amino acid, which carries a —SH moiety. Such a compound may also be anynon-amino compound or moiety, which contains or allows to introduce a—SH moiety into hydrophilic polymers P¹ and P³ as defined herein. Suchnon-amino compounds may be attached to the hydrophilic polymers P¹ andP³ of formula (VI) of the polymeric carrier according to the presentinvention via chemical reactions or binding of compounds, e.g. bybinding of a 3-thio propionic acid or thioimolane, by amide formation(e.g. carboxylic acids, sulphonic acids, amines, etc), by Michaeladdition (e.g maleinimide moieties, α,β unsatured carbonyls, etc), byclick chemistry (e.g. azides or alkines), by alkene/alkine methatesis(e.g. alkenes or alkines), imine or hydrozone formation (aldehydes orketons, hydrazins, hydroxylamins, amines), complexation reactions(avidin, biotin, protein G) or components which allow S_(n)-typesubstitution reactions (e.g halogenalkans, thiols, alcohols, amines,hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) orother chemical moieties which can be utilized in the attachment offurther components. A particularly preferred PEG derivate in thiscontext is alpha-Methoxy-omega-mercapto poly(ethylene glycol). In eachcase, the SH-moiety, e.g. of a cysteine or of any further (modified)amino acid or compound, may be present at the terminal ends orinternally at any position of hydrophilic polymers P¹ and P³. As definedherein, each of hydrophilic polymers P¹ and P³ typically exhibits atleast one —SH-moiety preferably at one terminal end, but may alsocontain two or even more —SH-moieties, which may be used to additionallyattach further components as defined herein, preferably furtherfunctional peptides or proteins e.g. a ligand, an amino acid component(AA) or (AA)_(x), antibodies, cell penetrating peptides or enhancerpeptides (e.g. TAT, KALA), etc.

According to one preferred alternative, such further functional peptidesor proteins may comprise so called cell penetrating peptides (CPPs) orcationic peptides for transportation. Particularly preferred are CPPs,which induce a pH-mediated conformational change in the endosome andlead to an improved release of the inventive polymeric carrier (incomplex with a nucleic acid) from the endosome by insertion into thelipid layer of the liposome. Such called cell penetrating peptides(CPPs) or cationic peptides for transportation, may include, withoutbeing limited thereto protamine, nucleoline, spermine or spermidine,poly-L-lysine (PLL), basic polypeptides, polyarginine, cell penetratingpeptides (CPPs), chimeric CPPs, such as Transportan, or MPG peptides,HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides,oligoarginines, members of the penetratin family, e.g. Penetratin,Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, pIsl, etc., antimicrobial-derived CPPs e.g.Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP,MAP, PpTG20, Proline-rich peptides, Loligomers, Arginine-rich peptides,Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine, poly-Arginine,histones, VP22 derived or analog peptides, Pestivirus Erns, HSV, VP22(Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.

According to a further preferred aspect of the first embodiment of thepresent invention, each of hydrophilic polymers P¹ and P³ of formula(VI) of the polymeric carrier used according to the present inventionmay also contain at least one further functional moiety, which allowsattaching further components as defined herein, e.g. a ligand as definedabove, or functionalities which allow the attachment of furthercomponents, e.g. by amide formation (e.g. carboxylic acids, sulphonicacids, amines, etc), by Michael addition (e.g maleinimide moieties, α,βunsatured carbonyls, etc), by click chemistry (e.g. azides or alkines),by alkene/alkine methatesis (e.g. alkenes or alkines), imine orhydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins,amines), complexation reactions (avidin, biotin, protein G) orcomponents which allow S_(e)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components. Furtherfunctional moieties may comprise an amino acid component (AA) as definedherein or (AA)_(x)., wherein (AA) is preferably an amino component asdefined above. In the above context, x is preferably an integer and maybe selected from a range of about 1 to 100, preferably from a range ofabout 1 to 50, more preferably 1 to 30, and even more preferablyselected from a number comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15-30, e.g. from a range of about 1 to 30, from a range ofabout 1 to 15, or from a number comprising 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15, or may be selected from a range formed by anytwo of the afore mentioned values. Most preferably, x is 1. Such anamino acid component (AA) or (AA)_(x) may be contained in every part ofthe inventive polymeric carrier according to formula (VI) above andtherefore may be attached to all components of the inventive polymericcarrier according to formula (VI). It is particularly preferred thatamino acid component (AA) or (AA)_(x) is present as a ligand or part ofthe repetitive component [S—P²—S]_(n) within formula (VI) of theinventive polymeric carrier.

In the context of the entire formula (VI) of the inventive polymericcarrier may be preferably defined as follows:L-P¹—S-[Cys-P²-Cys]_(n)-S—P³-Lwherein L, P¹, P², P³ and n are as defined herein, S is sulphur and eachCys provides for one —SH-moiety for the disulfide bond.

According to a particular aspect, the inventive polymeric carrieraccording to formula (VI) as defined above, may comprise at least oneamino acid component (AA) or (AA)_(x), as defined above. Such an aminoacid component (AA) or (AA)_(x) may be contained in every part of theinventive polymeric carrier according to formula (VI) above andtherefore may be attached to all components of the inventive polymericcarrier according to formula (VI). It is particularly preferred thatamino acid component (AA) or (AA)_(x) is present as a ligand or part ofthe repetitive component [S—P²—S]_(n) within formula (VI) of theinventive polymeric carrier. The amino acid component (AA) or (AA)_(x)preferably contains or is flanked (e.g. terminally) by at least one —SHcontaining moiety, which allows introducing this component (AA) or(AA)_(x) via a disulfide bond into the polymeric carrier according toformula (VI) as defined herein. Such a —SH-containing moiety may be any—SH containing moiety (or, of course, one sulphur of a disulfide bond),e.g. a cysteine residue. In the specific case that the —SH containingmoiety represents a cysteine, the amino acid component (AA)_(x) may alsobe read as -Cys-(AA)_(x)- or -Cys-(AA)_(x)-Cys-wherein Cys representsCysteine and provides for the necessary —SH-moiety for a disulfide bond.The —SH containing moiety may be also introduced into the amino acidcomponent (AA)_(x) using any of modifications or reactions as shownabove for components P¹, P² or P³. In the specific case that the aminoacid component (AA)_(x) is linked to two components of the inventivepolymeric carrier according to formula (VI) it is preferred that (AA) or(AA)_(x) contains at least two —SH-moieties, e.g. at least twoCysteines, preferably at its terminal ends. This is particularlypreferred if (AA) or (AA)_(x) is part of the repetitive component[S—P²—S]_(n). Alternatively, the amino acid component (AA) or (AA)_(x)is introduced into the inventive polymeric carrier according to formula(VI) as defined herein via any chemical possible addition reaction.Therefore the amino acid component (AA) or (AA)_(x) contains at leastone further functional moiety, which allows attaching same to a furthercomponent as defined herein, e.g. component P¹ or P³′ P², L, or afurther amino acid component (AA) or (AA)_(x), etc. Such functionalmoieties may be selected from functionalities which allow the attachmentof further components, e.g. functionalities as defined herein, e.g. byamide formation (e.g. carboxylic acids, sulphonic acids, amines, etc),by Michael addition (e.g maleinimide moieties, α,β unsatured carbonyls,etc), by click chemistry (e.g. azides or alkines), by alkene/alkinemethatesis (e.g. alkenes or alkines), imine or hydrozone formation(aldehydes or ketons, hydrazins, hydroxylamins, amines), complexationreactions (avidin, biotin, protein G) or components which allowS_(e)-type substitution reactions (e.g halogenalkans, thiols, alcohols,amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphoniumsalts) or other chemical moieties which can be utilized in theattachment of further components.

The amino acid component (AA) or (AA)_(x) in the polymeric carrier offormula (VI) may also occur as a mixed repetitive amino acid component[(AA)_(x)]_(z), wherein the number of amino acid components (AA) or(AA)_(x) is further defined by integer z. In this context, z may beselected from a range of about 1 to 30, preferably from a range of about1 to 15, more preferably 1 to 10 or 1 to 5 and even more preferablyselected from a number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15, or may be selected from a range formed by any two ofthe afore mentioned values.

According to a specific and particularly preferred alternative, theamino acid component (AA) or (AA)_(x), preferably written asS-(AA)_(x)-S or [S-(AA)_(x)-S] may be used to modify component P²,particularly the content of component S—P²—S in repetitive component[S—P²—S]_(n) of the polymeric carrier of formula (VI) above. This may berepresented in the context of the entire polymeric carrier according toformula (VI) e.g. by following formula (VIa):L-P¹—S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-L,wherein x, S, L, AA, P¹, P² and P³ are preferably as defined herein. Informula (VIa) above, any of the single components [S—P²—S] and[S-(AA)_(x)-S] may occur in any order in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]b}. The numbers of single components [S—P²—S]and [S-(AA)_(x)-S] in the subformula {[S—P²—S]_(a)[S-(AA)_(x)-S]b} aredetermined by integers a and b, wherein a+b=n. n is an integer and isdefined as above for formula (VI).

a is an integer, typically selected independent from integer b from arange of about 1 to 50, preferably from a range of about 1, 2 or 3 to30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or arange of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4,or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. arange of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range ofabout 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to11 or 7 to 10. Most preferably, a is in a range of about 1, 2, 3, 4, or5 to 10, more preferably in a range of about 1, 2, 3, or 4 to 9, in arange of about 1, 2, 3, or 4 to 8, or in a range of about 1, 2, or 3 to7.

b is an integer, typically selected independent from integer a from arange of about 0 to 50 or 1 to 50, preferably from a range of about 0,1, 2 or 3 to 30, more preferably from a range of about 0, 1, 2, 3, 4, or5 to 25, or a range of about 0, 1, 2, 3, 4, or 5 to 20, or a range ofabout 0, 1, 2, 3, 4, or 5 to 15, or a range of about 0, 1, 2, 3, 4, or 5to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or arange of about 6 to 11 or 7 to 10. Most preferably, b is in a range ofabout 1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2,3, or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range ofabout 1, 2, or 3 to 7.

According to one preferred aspect, the mRNA of the inventive vaccineencoding at least one antigen as defined above may be formulatedtogether with a cationic or polycationic compound and/or with apolymeric carrier, preferably as defined herein.

According to a further preferred aspect, the mRNA of the inventivevaccine encoding at least one antigen as defined above may be formulatedtogether with an (adjuvant) component. According to a particularlypreferred aspect, the mRNA of the inventive vaccine encoding at leastone antigen as defined above may be formulated to comprise a) an(adjuvant) component, comprising or consisting of at least oneimmunostimulatory nucleic acid, complexed with a cationic orpolycationic compound and/or with a polymeric carrier, preferably asdefined herein, and b) at least one free mRNA, encoding an antigen,preferably as defined herein for the inventive vaccine.

In the above context, a cationic or polycationic compound and/or apolymeric carrier used to complex the at least one immunostimulatorynucleic acid in the adjuvant component, may be selected from a cationicor polycationic compound and/or a polymeric carrier as defined above.

Furthermore, an immunostimulatory nucleic acid as defined above for theadjuvant component may be preferably selected from an mRNA as definedherein for the inventive vaccine, encoding at least one antigen.Alternatively, such an immunostimulatory nucleic acid may be selectedfrom an immunostimulatory nucleic acid, as defined herein, preferably animmunostimulatory RNA (isRNA) as defined herein.

In this context, an immunostimulatory nucleic acid, as used herein, ispreferably selected from immunostimulatory nucleic acids which are knownto bind to TLR receptors. Such an immunostimulatory nucleic acid can bein the form of a(n) (immunostimulatory) CpG nucleic acid, in particularCpG-RNA or CpG-DNA, which preferably induces an innate immune response.A CpG-RNA or CpG-DNA used according to the invention can be asingle-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA),a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid used according to the invention ispreferably in the form of CpG-RNA, more preferably in the form ofsingle-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG nucleicacids have a length as described above. Preferably the CpG motifs areunmethylated.

Furthermore, an immunostimulatory nucleic acid, as used herein, ispreferably selected from an immunostimulatory RNA (isRNA), whichpreferably elicits an innate immune response. Preferably, theimmunostimulatory RNA may be a single-stranded, a double-stranded or apartially double-stranded RNA, more preferably a single-stranded RNA,and/or a circular or linear RNA, more preferably a linear RNA. Morepreferably, the immunostimulatory RNA may be a (linear) single-strandedRNA. Even more preferably, the immunostimulatory RNA may be a (long)(linear) (single-stranded) non-coding RNA. In this context it isparticular preferred that the isRNA carries a triphosphate at its 5′-endwhich is the case for in vitro transcribed RNA. An immunostimulatory RNAmay also occur as a short RNA oligonucleotide as defined herein. Animmunostimulatory RNA as used herein may furthermore be selected fromany class of RNA molecules, found in nature or being preparedsynthetically, and which can induce an innate immune response and maysupport an adaptive immune response induced by an antigen. In thiscontext, an immune response may occur in various ways. A substantialfactor for a suitable (adaptive) immune response is the stimulation ofdifferent T cell sub-populations. T-lymphocytes are typically dividedinto two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2(Th2) cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of the effectorproteins (cytokines) produced by them. Thus, Th1 cells assist thecellular immune response by activation of macrophages and cytotoxic Tcells. Th2 cells, on the other hand, promote the humoral immune responseby stimulation of B cells for conversion into plasma cells and byformation of antibodies (e.g. against antigens). The Th1/Th2 ratio istherefore of great importance in the induction and maintenance of anadaptive immune response. In connection with the present invention, theTh1/Th2 ratio of the (adaptive) immune response is preferably shifted inthe direction towards the cellular response (Th1 response) and acellular immune response is thereby induced. According to one example,the innate immune system which may support an adaptive immune responsemay be activated by ligands of Toll-like receptors (TLRs). TLRs are afamily of highly conserved pattern recognition receptor (PRR)polypeptides that recognize pathogen-associated molecular patterns(PAMPs) and play a critical role in innate immunity in mammals.Currently at least thirteen family members, designated TLR1-TLR13(Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore,a number of specific TLR ligands have been identified. It was e.g. foundthat unmethylated bacterial DNA and synthetic analogs thereof (CpG DNA)are ligands for TLR9 (Hemmi H et al. (2000) Nature 408:740-5; Bauer S etal. (2001) Proc Natl. Acad. Sci. USA 98, 9237-42). Furthermore, it hasbeen reported that ligands for certain TLRs include certain nucleic acidmolecules and that certain types of RNA are immunostimulatory in asequence-independent or sequence-dependent manner, wherein these variousimmunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, orintracellular receptors such as RIG-I, MDA-5, etc.

Preferably, an immunostimulatory nucleic acid, preferably animmunostimulatory RNA (isRNA), as used herein, may comprise any RNAsequence known to be immunostimulatory, including, without being limitedthereto, RNA sequences representing and/or encoding ligands of TLRs,preferably selected from human family members TLR1-TLR10 or murinefamily members TLR1-TLR13, more preferably selected from (human) familymembers TLR1-TLR10, even more preferably from TLR7 and TLR8, ligands forintracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g.Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases:two parallel ways to trigger antiviral responses. Mol. Cell 22,561-569), or any other immunostimulatory RNA sequence. Furthermore,(classes of) immunostimulatory RNA molecules, used as a further compoundof the inventive vaccine, may include any other RNA capable of elicitingan immune response. Without being limited thereto, such animmunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA(tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such animmunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of5 to 50 or of 5 to 30 nucleotides.

According to a particularly preferred embodiment, an immunostimulatorynucleic acid sequence, particularly an isRNA, as used herein, mayconsist of or comprise a nucleic acid of formula (I) or (II):G_(l)X_(m)G_(n),  (formula (I))

-   wherein:-   G is guanosine, uracil or an analogue of guanosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the above-mentioned nucleotides;-   l is an integer from 1 to 40,    -   wherein    -   when l=1 G is guanosine or an analogue thereof,    -   when l>1 at least 50% of the nucleotides are guanosine or an        analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3 X is uracil or an analogue thereof,    -   when m>3 at least 3 successive uracils or analogues of uracil        occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1 G is guanosine or an analogue thereof,    -   when n>1 at least 50% of the nucleotides are guanosine or an        analogue thereof.        C_(l)X_(m)C_(n),  (formula (II))-   wherein:-   C is cytosine, uracil or an analogue of cytosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the above-mentioned nucleotides;-   l is an integer from 1 to 40,    -   wherein    -   when l=1 C is cytosine or an analogue thereof,    -   when l>1 at least 50% of the nucleotides are cytosine or an        analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3 X is uracil or an analogue thereof,    -   when m>3 at least 3 successive uracils or analogues of uracil        occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1 C is cytosine or an analogue thereof,    -   when n>1 at least 50% of the nucleotides are cytosine or an        analogue thereof.

The nucleic acids of formula (I) or (II), which may be used as animmunostimulatory nucleic acid sequence, particularly an isRNA, may berelatively short nucleic acid molecules with a typical length ofapproximately from 5 to 100 (but may also be longer than 100 nucleotidesfor specific embodiments, e.g. up to 200 nucleotides), from 5 to 90 orfrom 5 to 80 nucleotides, preferably a length of approximately from 5 to70, more preferably a length of approximately from 8 to 60 and, morepreferably a length of approximately from 15 to 60 nucleotides, morepreferably from 20 to 60, most preferably from 30 to 60 nucleotides. Ifthe nucleic acid of formula (I) or (II) has a maximum length of e.g. 100nucleotides, m will typically be <=98. The number of nucleotides G inthe nucleic acid of formula (I) is determined by 1 or n. 1 and n,independently of one another, are each an integer from 1 to 40, whereinwhen 1 or n=1 G is guanosine or an analogue thereof, and when 1 or n>1at least 50% of the nucleotides are guanosine or an analogue thereof.For example, without implying any limitation, when 1 or n=4 G_(l) orG_(n) can be, for example, a GUGU, GGUU, UGUG, UUGG, GUUG, GGGU, GGUG,GUGG, UGGG or GGGG, etc.; when 1 or n=5 G_(l) or G_(n) can be, forexample, a GGGUU, GGUGU, GUGGU, UGGGU, UGGUG, UGUGG, UUGGG, GUGUG,GGGGU, GGGUG, GGUGG, GUGGG, UGGGG, or GGGGG, etc.; etc. A nucleotideadjacent to X_(m) in the nucleic acid of formula (I) according to theinvention is preferably not a uracil. Similarly, the number ofnucleotides C in the nucleic acid of formula (II) according to theinvention is determined by l or n. l and n, independently of oneanother, are each an integer from 1 to 40, wherein when 1 or n=1 C iscytosine or an analogue thereof, and when 1 or n>1 at least 50% of thenucleotides are cytosine or an analogue thereof. For example, withoutimplying any limitation, when 1 or n=4, C_(l) or C_(n) can be, forexample, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC, CUCC, UCCC or CCCC,etc.; when l or n=5 C_(l) or C_(n) can be, for example, a CCCUU, CCUCU,CUCCU, UCCCU, UCCUC, UCUCC, UUCCC, CUCUC, CCCCU, CCCUC, CCUCC, CUCCC,UCCCC, or CCCCC, etc.; etc. A nucleotide adjacent to X_(m) in thenucleic acid of formula (II) according to the invention is preferablynot a uracil. Preferably, for formula (I), when l or n>1, at least 60%,70%, 80%, 90% or even 100% of the nucleotides are guanosine or ananalogue thereof, as defined above. The remaining nucleotides to 100%(when guanosine constitutes less than 100% of the nucleotides) in theflanking sequences G_(l) and/or G_(n) are uracil or an analogue thereof,as defined hereinbefore. Also preferably, 1 and n, independently of oneanother, are each an integer from 2 to 30, more preferably an integerfrom 2 to 20 and yet more preferably an integer from 2 to 15. The lowerlimit of 1 or n can be varied if necessary and is at least 1, preferablyat least 2, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10. Thisdefinition applies correspondingly to formula (II).

According to a particularly preferred embodiment, a nucleic acidaccording to any of formulas (I) or (II) above, which may be used as animmunostimulatory nucleic acid sequence, particularly an isRNA, may beselected from a sequence consisting or comprising any of the followingsequences:

(SEQ ID NO: 289) GGUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 290)GGGGGUUUUUUUUUUGGGGG; (SEQ ID NO: 291)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 292)GUGUGUGUGUGUUUUUUUUUUUUUUUUGUGUGUGUGUGU; (SEQ ID NO: 293)GGUUGGUUGGUUUUUUUUUUUUUUUUUGGUUGGUUGGUU; (SEQ ID NO: 294)GGGGGGGGGUUUGGGGGGGG; (SEQ ID NO: 295) GGGGGGGGUUUUGGGGGGGG;(SEQ ID NO: 296) GGGGGGGUUUUUUGGGGGGG; (SEQ ID NO: 297)GGGGGGGUUUUUUUGGGGGG; (SEQ ID NO: 298) GGGGGGUUUUUUUUGGGGGG;(SEQ ID NO: 299) GGGGGGUUUUUUUUUGGGGG; (SEQ ID NO: 300)GGGGGGUUUUUUUUUUGGGG; (SEQ ID NO: 301) GGGGGUUUUUUUUUUUGGGG;(SEQ ID NO: 302) GGGGGUUUUUUUUUUUUGGG; (SEQ ID NO: 303)GGGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 304) GGGGUUUUUUUUUUUUUUGG;(SEQ ID NO: 305) GGUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 306)GUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 307) GGGGGGGGGGUUUGGGGGGGGG;(SEQ ID NO: 308) GGGGGGGGGUUUUGGGGGGGGG; (SEQ ID NO: 309)GGGGGGGGUUUUUUGGGGGGGG; (SEQ ID NO: 310) GGGGGGGGUUUUUUUGGGGGGG;(SEQ ID NO: 311) GGGGGGGUUUUUUUUGGGGGGG; (SEQ ID NO: 312)GGGGGGGUUUUUUUUUGGGGGG; (SEQ ID NO: 313) GGGGGGGUUUUUUUUUUGGGGG;(SEQ ID NO: 314) GGGGGGUUUUUUUUUUUGGGGG; (SEQ ID NO: 315)GGGGGGUUUUUUUUUUUUGGGG; (SEQ ID NO: 316) GGGGGUUUUUUUUUUUUUGGGG;(SEQ ID NO: 317) GGGGGUUUUUUUUUUUUUUGGG; (SEQ ID NO: 318)GGGUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 319) GGUUUUUUUUUUUUUUUUUUGG;(SEQ ID NO: 320) GGGGGGGGGGGUUUGGGGGGGGGG; (SEQ ID NO: 321)GGGGGGGGGGUUUUGGGGGGGGGG; (SEQ ID NO: 322) GGGGGGGGGUUUUUUGGGGGGGGG;(SEQ ID NO: 323) GGGGGGGGGUUUUUUUGGGGGGGG; (SEQ ID NO: 324)GGGGGGGGUUUUUUUUGGGGGGGG; (SEQ ID NO: 325) GGGGGGGGUUUUUUUUUGGGGGGG;(SEQ ID NO: 326) GGGGGGGGUUUUUUUUUUGGGGGG; (SEQ ID NO: 327)GGGGGGGUUUUUUUUUUUGGGGGG; (SEQ ID NO: 328) GGGGGGGUUUUUUUUUUUUGGGGG;(SEQ ID NO: 329) GGGGGGUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 330)GGGGGGUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 331) GGGGUUUUUUUUUUUUUUUUGGGG;(SEQ ID NO: 332) GGGUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 333)GUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 334)GGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 335)GGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 336)GGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 337)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 338)GGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 339)GGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGG; (SEQ ID NO: 340)GGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGG; (SEQ ID NO: 341)GGGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGGG; (SEQ ID NO: 342)GGUUUGG; (SEQ ID NO: 343) GGUUUUGG; (SEQ ID NO: 344) GGUUUUUGG;(SEQ ID NO: 345) GGUUUUUUGG; (SEQ ID NO: 346) GGUUUUUUUGG;(SEQ ID NO: 347) GGUUUUUUUUGG; (SEQ ID NO: 348) GGUUUUUUUUUGG;(SEQ ID NO: 349) GGUUUUUUUUUUGG; (SEQ ID NO: 350) GGUUUUUUUUUUUGG;(SEQ ID NO: 351) GGUUUUUUUUUUUUGG; (SEQ ID NO: 352) GGUUUUUUUUUUUUUGG;(SEQ ID NO: 353) GGUUUUUUUUUUUUUUGG; (SEQ ID NO: 354)GGUUUUUUUUUUUUUUUGG; (SEQ ID NO: 355) GGGUUUGGG; (SEQ ID NO: 356)GGGUUUUGGG; (SEQ ID NO: 357) GGGUUUUUGGG; (SEQ ID NO: 358) GGGUUUUUUGGG;(SEQ ID NO: 359) GGGUUUUUUUGGG; (SEQ ID NO: 360) GGGUUUUUUUUGGG;(SEQ ID NO: 361) GGGUUUUUUUUUGGG; (SEQ ID NO: 362) GGGUUUUUUUUUUGGG;(SEQ ID NO: 363) GGGUUUUUUUUUUUGGG; (SEQ ID NO: 364) GGGUUUUUUUUUUUUGGG;(SEQ ID NO: 365) GGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 366)GGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUU UUUUGGG;(SEQ ID NO: 367) GGGUUUUUUUUUUUUUUUGGGGGGUUUUUUUUUUUUUUUGGG;(SEQ ID NO: 368) GGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGG G;(short GU-rich, SEQ ID NO: 369) GGUUUUUUUUUUUUUUUGGG or (SEQ ID NO: 370)CCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUU UUUUCCC(SEQ ID NO: 371) CCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCC C(SEQ ID NO: 372) CCCUUUUUUUUUUUUUUUCCCCCCUUUUUUUUUUUUUUUCCCor from a sequence having at least 60%, 70%, 80%, 90%, or even 95%sequence identity with any of these sequences.

According to a further particularly preferred embodiment, animmunostimulatory nucleic acid sequence, particularly an isRNA, as usedherein, may consist of or comprise a nucleic acid of formula (III) or(IV):(N_(u)G_(l)X_(m)G_(n)N_(v))_(a),  (formula (III))

-   wherein:-   G is guanosine (guanine), uridine (uracil) or an analogue of    guanosine (guanine) or uridine (uracil), preferably guanosine    (guanine) or an analogue thereof;-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine), or an analogue of these    nucleotides (nucleosides), preferably uridine (uracil) or an    analogue thereof;-   N is a nucleic acid sequence having a length of about 4 to 50,    preferably of about 4 to 40, more preferably of about 4 to 30 or 4    to 20 nucleic acids, each N independently being selected from    guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of these    nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein    -   when l=1, G is guanosine (guanine) or an analogue thereof,    -   when l>1, at least 50% of these nucleotides (nucleosides) are        guanosine (guanine) or an analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3, X is uridine (uracil) or an analogue thereof, and    -   when m>3, at least 3 successive uridines (uracils) or analogues        of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1, G is guanosine (guanine) or an analogue thereof,    -   when n>1, at least 50% of these nucleotides (nucleosides) are        guanosine (guanine) or an analogue thereof;-   u, v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v 1, or        -   when v=0, u≥1;            wherein the nucleic acid molecule of formula (III) has a            length of at least 50 nucleotides, preferably of at least            100 nucleotides, more preferably of at least 150            nucleotides, even more preferably of at least 200            nucleotides and most preferably of at least 250 nucleotides.            (N_(u)C_(l)X_(m)C_(n)N_(v))_(a)  (formula (IV))-   wherein:-   C is cytidine (cytosine), uridine (uracil) or an analogue of    cytidine (cytosine) or uridine (uracil), preferably cytidine    (cytosine) or an analogue thereof;-   X is guanosine (guanine), uridine (uracil), adenosine (adenine),    thymidine (thymine), cytidine (cytosine) or an analogue of the    above-mentioned nucleotides (nucleosides), preferably uridine    (uracil) or an analogue thereof;-   N is each a nucleic acid sequence having independent from each other    a length of about 4 to 50, preferably of about 4 to 40, more    preferably of about 4 to 30 or 4 to 20 nucleic acids, each N    independently being selected from guanosine (guanine), uridine    (uracil), adenosine (adenine), thymidine (thymine), cytidine    (cytosine) or an analogue of these nucleotides (nucleosides);-   a is an integer from 1 to 20, preferably from 1 to 15, most    preferably from 1 to 10;-   l is an integer from 1 to 40,    -   wherein    -   when 1=1, C is cytidine (cytosine) or an analogue thereof,    -   when 1>1, at least 50% of these nucleotides (nucleosides) are        cytidine (cytosine) or an analogue thereof;-   m is an integer and is at least 3;    -   wherein    -   when m=3, X is uridine (uracil) or an analogue thereof,    -   when m>3, at least 3 successive uridines (uracils) or analogues        of uridine (uracil) occur;-   n is an integer from 1 to 40,    -   wherein    -   when n=1, C is cytidine (cytosine) or an analogue thereof,    -   when n>1, at least 50% of these nucleotides (nucleosides) are        cytidine (cytosine) or an analogue thereof.-   u, v may be independently from each other an integer from 0 to 50,    -   preferably wherein when u=0, v 1, or        -   when v=0, u≥1;            wherein the nucleic acid molecule of formula (IV) according            to the invention has a length of at least 50 nucleotides,            preferably of at least 100 nucleotides, more preferably of            at least 150 nucleotides, even more preferably of at least            200 nucleotides and most preferably of at least 250            nucleotides.

Any of the definitions given above in formulae (I) and (II), e.g. forelements N (i.e. N_(u) and N_(v)) and X (X_(m)), particularly the corestructure as defined above, as well as for integers a, 1, m, n, u and v,similarly apply to elements of formula (III) and (IV) correspondingly.The definition of bordering elements N_(u) and N_(v) in formula (IV) isidentical to the definitions given above for N_(u) and N_(v) in formula(IV).

According to a very particularly preferred embodiment, the inventivenucleic acid molecule according to formula (IV), which may be used as animmunostimulatory nucleic acid sequence, particularly an isRNA, may beselected from e.g. any of the following sequences:

(SEQ ID NO: 373) UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACG (SEQ ID NO: 374)UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUCCAAAAAAAAAAAAAAACCC ACGCAAGGAUCUUCAUGUGC(SEQ ID NO: 375) GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUC (SEQ ID NO: 376)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAG (SEQ ID NO: 377)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAGAGCUACGCAGGUUCGCAAUAAAAGCGUUGAUUAGUGUGCAUAGAACAGACCUCUUAUUCGGUGAAACGCCAGAAUGCUAAAUUCCAAUAACUCUUCCCAAAACGCGUACGGCCGAAGACGCGCGCUUAUCUUGUGUACGUUCUCGCACAUGGAAGAAUCAGCGGGCAUGGUGGUAGGGCAAUAGGGGAGCUGGGUAGCAGCGAAAAAGGGCCCCUGCGCACGUAGCUUCGCUGUUCGUCUGAAACAACCCGGCAUCCGUUGUAGCGAUCCCGUUAUCAGUGUUAUUCUUGUGCGCACUAAGAUUCAUGGUGUAGUCGACAAUAACAGCGUCUUGGCAGAUUCUGGUCACGUGCCCUAUGCCCGGGCUUGUGCCUCUCAGGUGCACAGCGAUACUUAAAGCCUUCAAGGUACUCGACGUGGGUACCGAUUCGUGACACUUCCUAAGAUUAUUCCACUGUGUUAGCCCCGCACCGCCGACCUAAACUGGUCCAAUGUAUACGCAUUCGCUGAGCGGAUCGAUAAUAAAAGCUUGAAUU (SEQ ID NO: 378)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUC (R 722 SEQ ID NO: 379)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUA  (SEQ ID NO: 380)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAGAACGAACUGACCUGACGCCUGAACUUAUGAGCGUGCGUAUUUUUUUUUUUUUUUUUUUUUUUCCUCCCAACAAAUGUCGAUCAAUAGCUGGGCUGUUGGAGACGCGUCAGCAAAUGCCGUGGCUCCAUAGGACGUGUAGACUUCUAUUUUUUUUUUUUUUUUUUUUUCCCGGGACCACAAAUAAUAUUCUUGCUUGGUUGGGCGCAAGGGCCCCGUAUCAGGUCAUAAACGGGUACAUGUUGCACAGGCUCCUUUUUUUUUUUUUUUUUUUUUUUCGCUGAGUUAUUCCGGUCUCAAAAGACGGCAGACGUCAGUCGACAACACGGUCUAAAGCAGUGCUACAAUCUGCCGUGUUCGUGUUUUUUUUUUUUUUUUUUUUGUGAACCUACACGGCGUGCACUGUAGUUCGCAAUUCAUAGGGUACCGGCUCAGAGUUAUGCCUUGGUUGAAAACUGCCCAGCAUACUUUUUUUUUUUUUUUUUUUUCAUAUUCCCAUGCUAAGCAAGGGAUGCCGCGAGUCAUGUUAAGCUUGAAUU

According to another very particularly preferred embodiment, the nucleicacid molecule according to formula (V) may be selected from e.g. any ofthe following sequences:

(SEQ ID NO: 381) UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACG or (SEQ ID NO: 382)UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUGGAAAAAAAAAAAAAAAGGG ACGCAAGGAUCUUCAUGUGCor from a sequence having at least 60%, 70%, 80%, 90%, or even 95%sequence identity with any of these sequences.

Finally, the so called “(adjuvant) component”, which may be used totogether with the mRNA in the inventive vaccine, is preferably preparedaccording to a first step by complexing the at least one (m)RNA of the(adjuvant) component with a cationic or polycationic compound and/orwith a polymeric carrier, preferably as defined herein, in a specificratio to form a stable complex. In this context, it is highlypreferable, that no free cationic or polycationic compound or polymericcarrier or only a neclectably small amount thereof remains in the(adjuvant) component after complexing the (m)RNA. Accordingly, the ratioof the (m)RNA and the cationic or polycationic compound and/or thepolymeric carrier in the (adjuvant) component is typically selected in arange that the (m)RNA is entirely complexed and no free cationic orpolycationic compound or polymeric carrier or only a neclectably smallamount thereof remains in the composition. Preferably the ratio of the(adjuvant) component, i.e. the ratio of the (m)RNA to the cationic orpolycationic compound and/or the polymeric carrier, preferably asdefined herein, is selected from a range of about 6:1 (w/w) to about0,25:1 (w/w), more preferably from about 5:1 (w/w) to about 0,5:1 (w/w),even more preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1(w/w) to about 2:1 (w/w). Alternatively, the ratio of the (m)RNA to thecationic or polycationic compound and/or the polymeric carrier,preferably as defined herein, in the (adjuvant) component, may also becalculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) ofthe entire complex of the (adjuvant) component. In the context of thepresent invention, an N/P-ratio is preferably in the range of about0.1-10, preferably in a range of about 0.3-4 and most preferably in arange of about 0.5-2 or 0.7-2 regarding the ratio of (m)RNA: cationic orpolycationic compound and/or polymeric carrier, preferably as definedherein, in the complex, and most preferably in the range of about0.7-1.5, preferably provided the cationic or polycationic compound inthe complex is a cationic or polycationic cationic or polycationicprotein or peptide and/or the polymeric carrier is as defined above.Such ratios, particularly weight and/or N/P ratios may also be appliedto ratios of the at least one mRNA encoding at least one antigen asdefined herein to a cationic or polycationic polymer or a polymericcarrier as defined herein used to complex the at least one mRNA.

According to a further preferred aspect, the mRNA of the inventivevaccine encoding at least one antigen as defined above may be formulatedtogether with an (adjuvant) component as defined above, wherein theinventive vaccine may comprise a) an (adjuvant) component, comprising orconsisting of at least one (m)RNA, complexed with a cationic orpolycationic compound and/or with a polymeric carrier, preferably asdefined herein, and b) at least one free mRNA, encoding an antigen,preferably as defined herein. This formulation is preferably as definedabove. Furthermore, the entire formulation of a) and b) may beadditionally packaged with a carrier molecule to allow combinedpackaging the (adjuvant) component and the antigen. Such a carriermolecule may be selected from any polymer suitable for packaging andpreferably transporting the entire formulation of a) and b) into cells,tissue, etc., of a patient as defined herein, e.g. from a cationic orpolycationic polymer as defined herein or from any further polymersuitable for this purpose, e.g. a polymeric carrier as defined above.

The ratio of all components of the entire inventive vaccine composition,as defined above, preferably, an adjuvant component comprising orconsisting of at least one immunostimulatory nucleic acid sequence,complexed with a cationic or polycationic compound, the at least onemRNA encoding at least one antigen, and/or a carrier molecule,formulated in the inventive vaccine, may be calculated on the basis ofthe nitrogen/phosphate ratio (N/P-ratio) of all these components. In thecontext of the present invention, an N/P-ratio is preferably in therange of about 0.01-4, 0.01-2, 0.1-2 or 0.1-1.5 regarding the ratio ofnucleic acids:cationic or polycationic peptide contained in theinventive vaccine, and most preferably in the range of about 0.1-1. Suchan N/P ratio is preferably designed to provide good transfectionproperties in vivo and transport into and through cell membranes.Preferably, for this purpose, cationic or polycationic compound and/orpolymeric carriers as used herein, are based on peptide sequences.

In a further preferred aspect of the present invention the inventivevaccine may comprise a pharmaceutically acceptable carrier and/orvehicle. In the context of the present invention, a pharmaceuticallyacceptable carrier typically includes the liquid or non-liquid basis ofa composition comprising the components of the inventive vaccine. If thecomposition is provided in liquid form, the carrier will typically bepyrogen-free water; isotonic saline or buffered (aqueous) solutions,e.g. phosphate, citrate etc. buffered solutions. The injection buffermay be hypertonic, isotonic or hypotonic with reference to the specificreference medium, i.e. the buffer may have a higher, identical or lowersalt content with reference to the specific reference medium, whereinpreferably such concentrations of the afore mentioned salts may be used,which do not lead to damage of cells due to osmosis or otherconcentration effects. Reference media are e.g. liquids occurring in “invivo” methods, such as blood, lymph, cytosolic liquids, or other bodyliquids, or e.g. liquids, which may be used as reference media in “invitro” methods, such as common buffers or liquids. Such common buffersor liquids are known to a skilled person. Ringer-Lactate solution isparticularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds, which are suitable for administration to apatient to be treated, may be used as well for the inventive vaccine.The term “compatible” as used here means that these constituents of theinventive vaccine are capable of being mixed with the components of theinventive vaccine in such a manner that no interaction occurs whichwould substantially reduce the pharmaceutical effectiveness of theinventive vaccine under typical use conditions.

According to a specific aspect, the inventive vaccine may comprise anadjuvant. In this context, an adjuvant may be understood as anycompound, which is suitable to initiate or increase an immune responseof the innate immune system, i.e. a non-specific immune response. Withother words, when administered, the vaccine preferably elicits an innateimmune response due to the adjuvant, optionally contained therein.Preferably, such an adjuvant may be selected from an adjuvant known to askilled person and suitable for the present case, i.e. supporting theinduction of an innate immune response in a mammal, e.g. an adjuvantprotein as defined above or an adjuvant as defined in the following.

According to one aspect such an adjuvant may be selected from an(adjuvant) component as defined above.

According to one further aspect such an adjuvant may be selected fromany adjuvant known to a skilled person and suitable for the presentcase, i.e. supporting the induction of an innate immune response in amammal and/or suitable for depot and delivery of the components of theinventive vaccine. Preferred as adjuvants suitable for depot anddelivery are cationic or polycationic compounds as defined above.Likewise, the adjuvant may be selected from the group consisting of,without being limited thereto, cationic or polycationic compounds asdefined above, from chitosan, TDM, MDP, muramyl dipeptide, pluronics,alum solution, aluminium hydroxide, ADJUMER™ (polyphosphazene);aluminium phosphate gel; glucans from algae; algammulin; aluminiumhydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel;low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane(5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline,pH 7.4); AVRIDINE™ (propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylaminob-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo [4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferongamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; IS COPREP7.0.3.™; liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT 5 oraladjuvant (E. coli labile enterotoxin-protoxin); microspheres andmicroparticles of any composition; MF59™; (squalenewater emulsion);MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA720™ (metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryllipid A); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-AlaD-Gln-OCH-3); MURAPALMITINE™and DMURAPALMITINE™ (Nac-Mur-L-Thr-DisoGln-sn-glyceroldipalmitoyl); NAGO(neuraminidase-galactose oxidase); nanospheres or nanoparticles of anycomposition; NISVs (non-ionic surfactant vesicles); PLEURAN™ (□-glucan);PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolicacid; microspheres/nanospheres); PLURONIC L121™; PMMA(polymethylmethacrylate); PODDS™ (proteinoid microspheres); polyethylenecarbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylicacid complex); polysorbate 80 (Tween 80); protein cochleates (AvantiPolar Lipids, Inc., Alabaster, Ala.); STIMULON™ (QS-21); Quil-A (Quil-Asaponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”);Sendai proteoliposomes and Sendaicontaining lipid matrices; Span-85(sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween85); squalene or ROBANE® (2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); THERAMID®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-LAladipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;Nacetylmuramyl-Lthreonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin, microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, 35 IC31, Imidazoquinolines, Ampligen, Ribi529,IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys,Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides,UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable asantagonists including CGRP neuropeptide.

Particularly preferred, an adjuvant may be selected from adjuvants,which support induction of a Th1-immune response or maturation of naïveT cells, such as GM-CSF, IL-12, IFNγ, immunostimulatory RNA sequences asdefined herein, CpG DNA, etc.

The inventive vaccine may additionally contain a furtherimmunotherapeutic agent selected from immunoglobulins, preferably IgGs,monoclonal or polyclonal antibodies, polyclonal serum or sera, etc.Preferably, such a further immunotherapeutic agent may be provided as apeptide/protein or may be encoded by a nucleic acid, preferably by a DNAor an RNA, more preferably an mRNA. Such an immunostimulatory agentallows providing passive vaccination additional to active vaccinationtriggered by the mRNA encoded antigen of the inventive composition orvaccine composition.

The inventive vaccine can additionally contain one or more auxiliarysubstances in order to increase its immunogenicity or immunostimulatorycapacity, if desired. A synergistic action of the inventive vaccine andof an auxiliary substance, which may be optionally contained in thevaccine or may be formulated with the inhibitor, is preferably achievedthereby. Depending on the various types of auxiliary substances, variousmechanisms can come into consideration in this respect. For example,compounds that permit the maturation of dendritic cells (DCs), forexample lipopolysaccharides, TNF-alpha or CD40 ligand, form a firstclass of suitable auxiliary substances. In general, it is possible touse as auxiliary substance any agent that influences the immune systemin the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, suchas GM-CFS, which allow an immune response to be enhanced and/orinfluenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that further promote the innate immune response, such asIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32,IL-33, IFNα, IFNβ, IFNγ, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha,growth factors, such as hGH.

The inventive vaccine can also additionally contain any furthercompound, which is known to be immunostimulating due to its bindingaffinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, aligand of a NOD-like receptor, or a ligand of a RIG-I like receptor.

In this context the inventive vaccine may also additionally contain animmunostimulatory nucleic acid, preferably an immunostimulatory RNA(isRNA), as defined above.

The inventive vaccine as defined according to the first embodiment ofthe present invention may furthermore comprise further additives oradditional compounds. Further additives which may be included in theinventive vaccine are emulsifiers, such as, for example, TWEEN®; wettingagents, such as, for example, sodium lauryl sulfate; colouring agents;taste-imparting agents, pharmaceutical carriers; tablet-forming agents;stabilizers; antioxidants; preservatives.

One further additive, which may be contained in the inventive vaccine,may be an antibacterial agent. In this context, any anti-bacterialagents known to one of skill in the art may be used in combination withthe components of the inventive vaccine as defined herein. Non-limitingexamples of anti-bacterial agents include Amikacin, Amoxicillin,Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin,Ampicillin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin,Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin,Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime,Cefoperazone, Cefoperazonesulbactam, Cefotaxime, Cefoxitin, Cefbirome,Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam,Cefbrozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole,Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin,Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin,Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin,Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin,Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin,Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid,Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem,Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin,Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem,Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline,Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin,Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin,Penicillin V, Piperacillin, Piperacillin-sulbactam,Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin,Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole,Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin,Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin,Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, andVoriconazole.

Another additive, which may be contained in the inventive vaccine, maybe an anti-viral agents, preferably, but are not limited to, nucleosideanalogs (e.g., zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine,peramivir, rimantadine, saquinavir, indinavir, ritonavir,alpha-interferons and other interferons, AZT, t-705, zanamivir(RELENZA®), and oseltamivir (TAMIFLU®). Other anti-viral agents includeinfluenza virus vaccines, e.g., FLUARIX® (Glaxo SmithKline), FLUMIST®(MedImmune Vaccines), FLUVIRIN® (Chiron Corporation), FLULAVAL®(GlaxoSmithKline), AFLURIA® (CSL Biotherapies Inc.), AGRIFLU® (Novartis)or FLUZONE® (Aventis Pasteur).

The inventive vaccine typically comprises a “safe and effective amount”of the components of the inventive vaccine as defined herein. As usedherein, a “safe and effective amount” preferably means an amount of thecomponents, preferably of the at least one mRNA, that is sufficient tosignificantly induce a positive modification of a disease or disorder asdefined herein. At the same time, however, a “safe and effective amount”is small enough to avoid serious side-effects and to permit a sensiblerelationship between advantage and risk. The determination of theselimits typically lies within the scope of sensible medical judgment.

As defined according to the first embodiment, the inventive vaccinecomprising at least one mRNA encoding at least one antigen may be usedin the prophylaxis and treatment of a disease in newborns and/orinfants, preferably exhibiting an age of not more than 3 or 2 years,preferably of not more than 1 year (12 months), more preferably of notmore than 9, 6 or 3 months. The treatment preferably comprisesvaccination of the newborn or infant and eliciting an immune response insaid newborn or infant. Preferably, a newborn or infant is a mammal(patient), preferably a human (patient), typically exhibiting an age ofnot more than 3 or usually 2 years, preferably of not more than 1.5years, more preferably of not more than 1 year (12 months), even morepreferably of not more than 9 months, 6 months or even 3 months.Accordingly, a newborn or infant may comprise an age of about 0 to 3 orusually 0 to 2 years, preferably of 0 to 1.5 years, more preferably of 0to 1 years (0 to 12 months), even more preferably of not more than 0 to9 months, 0 to 6 months or even 0 to 3 months. A newborn or infant mayfurthermore be distinguished into a newborn, typically exhibiting an ageof not more than 1 year (12 months), preferably of not more than 9months, 6 months or even 3 months. Accordingly, a newborn or infant maycomprise an age of about 0 to 1 years (0 to 12 months), preferably ofnot more than 0 to 9 months, 0 to 6 months or even 0 to 3 months. Anewborn or infant may furthermore be distinguished into an infant,typically exhibiting an age of more than 3 months, preferably exhibitingan age of 6 months, more preferably exhibiting an age of more than 9months, but additionally exhibiting an age of not more than 3 or usually2 years, preferably of not more than 1.5 years, more preferably of notmore than 1 year (12 months), even more preferably of not more than 9months or even 6 months. Accordingly, a newborn or infant may comprisean age of about 3 months to about 3 years, about 3 months to about 2years, about 3 months to about 1.5 years, or about 3 months to about 1year (12 months), about 6 months to about 3 years, about 6 months toabout 2 years, about 6 months to about 1.5 years, or about 6 months toabout 1 year (12 months), about 9 months to about 3 years, about 9months to about 2 years, or about 9 months to about 1.5 years, about 12months to about 3 years, about 12 months to about 2 years, or about 12months to about 1.5 years. The newborns and or infant may be male orfemale.

As furthermore defined in the first embodiment of the present invention,the treatment comprises vaccination of the patient and eliciting animmune response in said patient. In this context, vaccination typicallyoccurs via administration of the inventive vaccine. Administration mayoccur parenterally, orally, nasally, pulmonary, by inhalation (e.g. viaan aerosol or spray), topically, rectally, buccally, vaginally, or viaan implanted reservoir. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional,intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal,intracardial, intraarterial, and sublingual injection or infusiontechniques. Preferably, the inventive vaccine may be administeredintradermally to reach APCs in the dermis. Likewise preferably, theinventive vaccine as defined herein may be administered orally in anyorally acceptable dosage form including, but not limited to, capsules,tablets, aqueous suspensions or solutions. Likewise preferably, theinventive vaccine may be administered topically, especially when thetarget of treatment includes areas or organs readily accessible bytopical application, e.g. including diseases of the skin or of any otheraccessible epithelial tissue. Suitable topical formulations are readilyprepared for each of these areas or organs. For topical applications,the inventive vaccine may be formulated in a suitable ointment,containing the inventive vaccine and optionally further components asdefined herein suspended or dissolved in one or more carriers. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent for use as aspray.

The inventive vaccine may be used in combination with other therapies,preferably with a therapy for a disease as defined herein, or furthertherapies. As used herein, the term “in combination,” in the context ofthe administration of two or more therapies to a newborn or an infant asdefined herein, refers to the use of more than one therapy, preferablytwo therapies or even more. The use of the term “in combination” doesnot restrict the order in which therapies are administered to a newbornor an infant as defined herein. For example, a first therapy (e.g., afirst prophylactic or therapeutic agent) can be administered at any timeprior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g.,5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksafter) the administration of a second therapy to a newborn or an infantas defined herein. In some aspects, the one or more other therapies aresurgery, immunotherapies, gene therapies, pain treatments, anti-fevermedications, therapies that alleviate or assist with breathing, other(active or passive) vaccinations/immunizations, antiviral therapies,antibacterial therapies, antifungal therapies, anti-parasite therapies,anti-allergic therapies, conventional tumour therapies, chemotherapies,or may include a post exposition prophylaxis in or for any of thediseases mentioned herein, preferably for rabies, RSV viral infection,etc.

In certain aspects, the therapies are administered less than 5 minutesapart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart,at about 1 to about 2 hours apart, at about 2 hours to about 3 hoursapart, at about 3 hours to about 4 hours apart, at about 4 hours toabout 5 hours apart, at about 5 hours to about 6 hours apart, at about 6hours to about 7 hours apart, at about 7 hours to about 8 hours apart,at about 8 hours to about 9 hours apart, at about 9 hours to about 10hours apart, at about 10 hours to about 11 hours apart, at about 11hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96hours apart, or 96 hours to 120 hours apart. In specific aspects, two ormore therapies are administered within the same patient visit.

Exemplary doses for mRNAs encoding at least one antigen as definedherein may range, without being limited thereto, from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 μg, or 30-300 μg mRNA per patient.Preferably, the inventive vaccine is formulated accordingly to compriseone dose, two doses, three or even more doses.

According to a specific aspect, the inventive vaccine may beadministered to a newborn or an infant as a single dose. In certainaspects, the inventive vaccine may be administered to a newborn or aninfant as a single dose followed by a second dose later and optionallyeven a third, fourth (or more) dose subsequent thereto etc. Inaccordance with this aspect, booster inoculations with the inventivevaccine may be administered to a newborn or an infant at specific timeintervals, preferably as defined below, following the second (or third,fourth, etc.) inoculation. In certain aspects, such booster inoculationswith the inventive vaccine may utilize an additional compound orcomponent as defined for the inventive vaccine as defined herein. Insome aspects, the administration of the same inventive vaccine and/orbooster administrations may be repeated and such administrations may beseparated by at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15days, 30 days, 45 days, 2 months, 75 days, e.g. 1 to 5 days, 1 to 10days, 5 to 15 days, 10 to 20 days, 15 to 25 days, 20 to 30 days, 25 to35 days, 30 to 50 days, 40 to 60 days, 50 to 70 days, 1 to 75 days, or 1month, 2 months, 3 months, 4 months, 5 months, or at least 6, 7, 8, 9,10, 11, 12 months, 18 months, 24 months, 30 months, 36 months, 1 year, 2years, 3 years, 5 years, 10 years, 15 years, 20 years, 30 years, 40years, 50 years, 60 years, or even more. In certain aspects, theinventive vaccine may be administered to a subject as a single dose onceper year.

In particular aspects, the inventive vaccine may be administered to anewborn or an infant in the fall or winter, i.e., prior to or during theinfluenza season in each hemisphere. In one aspect, a newborn or aninfant is administered his/her first dose early in the season, e.g.,late September or early October, so that the second dose (if necessary)can be given prior to the peak of the influenza season.

In particular aspects, the inventive vaccine may be administered atleast once, preferably twice or more to a newborn or an infant prior toa treatment of a disease as defined herein, preferably at least 1 day, 2days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2months, 75 days, e.g. 1 to 5 days, 1 to 10 days, 5 to 15 days, 10 to 20days, 15 to 25 days, 20 to 30 days, 25 to 35 days, 30 to 50 days, 40 to60 days, 50 to 70 days, 1 to 75 days, or 1 month, 2 months, 3 months, 4months, 5 months, or at least 6, 7, 8, 9, 10, 11, or 12 months prior toa treatment of a disease as defined herein. A second or further dose maythen be administered directly prior to treatment, concurrent with orsubsequent to treatment.

Furthermore, a disease as defined according to the first embodiment ofthe present invention is any disease selected from infectious diseases,preferably (viral, bacterial or protozoological) infectious diseases,autoimmune diseases, allergies or allergic diseases or cancer or tumourdiseases diseases.

Such diseases include cancer or tumour diseases, preferably selectedfrom melanomas, malignant melanomas, colon carcinomas, lymphomas,sarcomas, blastomas, renal carcinomas, gastrointestinal tumours,gliomas, prostate tumours, bladder cancer, rectal tumours, stomachcancer, oesophageal cancer, pancreatic cancer, liver cancer, mammarycarcinomas (=breast cancer), uterine cancer, cervical cancer, acutemyeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloidleukaemia (CML), chronic lymphocytic leukaemia (CLL), leukaemia,hepatomas, various virus-induced tumours such as, for example, papillomavirus-induced carcinomas (e.g. cervical carcinoma=cervical cancer),adenocarcinomas, herpes virus-induced tumours (e.g. Burkitt's lymphoma,EBV-induced B cell lymphoma), heptatitis B-induced tumours (hepatocellcarcinomas), HTLV-1- and HTLV-2-induced lymphomas, acoustic neuroma,lung carcinomas (=lung cancer=bronchial carcinoma), small-cell lungcarcinomas, pharyngeal cancer, anal carcinoma, glioblastoma, rectalcarcinoma, astrocytoma, brain tumours, retinoblastoma, basalioma, brainmetastases, medulloblastomas, vaginal cancer, pancreatic cancer,testicular cancer, Hodgkin's syndrome, meningiomas, Schneebergerdisease, hypophysis tumour, Mycosis fungoides, carcinoids, neurinoma,spinalioma, Burkitt's lymphoma, laryngeal cancer, renal cancer, thymoma,corpus carcinoma, bone cancer, non-Hodgkin's lymphomas, urethral cancer,CUP syndrome, head/neck tumours, oligodendroglioma, vulval cancer,intestinal cancer, colon carcinoma, oesophageal carcinoma (=oesophagealcancer), wart involvement, tumours of the small intestine,craniopharyngeomas, ovarian carcinoma, genital tumours, ovarian cancer(=ovarian carcinoma), pancreatic carcinoma (=pancreatic cancer),endometrial carcinoma, liver metastases, penile cancer, tongue cancer,gall bladder cancer, leukaemia, plasmocytoma, lid tumour, prostatecancer (=prostate tumours), etc.

According to one further specific aspect, diseases as defined hereincomprise infectious diseases, preferably (viral, bacterial orprotozoological) infectious diseases. Such infectious diseases,preferably viral, bacterial or protozoological infectious diseases, aretypically selected from viral infectious diseases such as influenza,preferably influenza-A, influenza-B, influenza-C or thogotovirus, morepreferably influenza-A comprising e.g. haemagglutinin subtypes H1, H2,H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14 or H15, and/orneuroamidase subtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9, orpreferably influenza-A subtypes H1N1, H1N2, H2N2, H2N3, H3N1, H3N2,H3N3, H5N1, H5N2, H7N7 or H9N2, etc., or any further combination,malaria, severe acute respiratory syndrome (SARS), respiratory syncytialvirus infection, yellow fever, AIDS, Lyme borreliosis, Leishmaniasis,anthrax, meningitis, Condyloma acuminata, hollow warts, Dengue fever,three-day fever, Ebola virus, cold, early summer meningoencephalitis(FSME), shingles, hepatitis, herpes simplex type I, herpes simplex typeII, Herpes zoster, Japanese encephalitis, Arenavirus-associated diseases(Lassa fever infection), Marburg virus, measles, foot-and-mouth disease,mononucleosis infectiosa (Pfeiffer's glandular fever), mumps, Norwalkvirus infection, smallpox, polio (childhood lameness), pseudo-croup,Erythema infectiosum (fifth disease), rabies, warts, West Nile fever,chickenpox, cytomegalic virus (CMV), bacterial infectious diseases suchas miscarriage (prostate inflammation), anthrax, appendicitis,borreliosis, botulism, Camphylobacter, Chlamydia trachomatis(inflammation of the urethra, conjunctivitis), cholera, diphtheria,donavanosis, epiglottitis, typhus fever, gas gangrene, gonorrhoea,rabbit fever, Heliobacter pylori, whooping cough, climatic bubo,osteomyelitis, Legionnaire's disease, leprosy, listeriosis, pneumonia,meningitis, bacterial meningitis, anthrax, otitis media, Mycoplasmahominis, neonatal sepsis (Chorioamnionitis), noma, paratyphus, plague,Reiter's syndrome, Rocky Mountain spotted fever, Salmonella paratyphus,Salmonella typhus, scarlet fever, syphilis, tetanus, tripper,tsutsugamushi disease, tuberculosis, typhus, vaginitis (colpitis), softchancre, and infectious diseases caused by parasites, protozoa or fungi,such as amoebiasis, bilharziosis, Chagas disease, Echinococcus, fishtapeworm, fish poisoning (Ciguatera), fox tapeworm, athlete's foot,canine tapeworm, candidosis, yeast fungus spots, scabies, cutaneousLeishmaniosis, lambliasis (giardiasis), lice, malaria, microscopy,onchocercosis (river blindness), fungal diseases, bovine tapeworm,schistosomiasis, porcine tapeworm, toxoplasmosis, trichomoniasis,trypanosomiasis (sleeping sickness), visceral Leishmaniosis,nappy/diaper dermatitis or miniature tapeworm.

According to another specific aspect, diseases as defined hereincomprise autoimmune diseases as defined in the following. Autoimmunediseases can be broadly divided into systemic and organ-specific orlocalised autoimmune disorders, depending on the principalclinico-pathologic features of each disease. Autoimmune diseases may bedivided into the categories of systemic syndromes, including systemiclupus erythematosus (SLE), Sjögren's syndrome, Scleroderma, RheumatoidArthritis and polymyositis or local syndromes which may beendocrinologic (type I diabetes (Diabetes mellitus Type 1), Hashimoto'sthyroiditis, Addison's disease etc.), dermatologic (pemphigus vulgaris),haematologic (autoimmune haemolytic anaemia), neural (multiplesclerosis) or can involve virtually any circumscribed mass of bodytissue. The autoimmune diseases to be treated may be selected from thegroup consisting of type I autoimmune diseases or type II autoimmunediseases or type III autoimmune diseases or type IV autoimmune diseases,such as, for example, multiple sclerosis (MS), rheumatoid arthritis,diabetes, type I diabetes (Diabetes mellitus Type 1), chronicpolyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis,colitis ulcerosa, type I allergy diseases, type II allergy diseases,type III allergy diseases, type IV allergy diseases, fibromyalgia, hairloss, Bechterew's disease, Crohn's disease, Myasthenia gravis,neurodermitis, Polymyalgia rheumatica, progressive systemic sclerosis(PSS), Reiter's syndrome, rheumatic arthritis, psoriasis, vasculitis,etc, or type II diabetes. While the exact mode as to why the immunesystem induces an immune reaction against autoantigens has not beenelucidated so far, there are several findings with regard to theetiology. Accordingly, the autoreaction may be due to a T cell bypass. Anormal immune system requires the activation of B cells by T cellsbefore the former can produce antibodies in large quantities. Thisrequirement of a T cell can be by-passed in rare instances, such asinfection by organisms producing super-antigens, which are capable ofinitiating polyclonal activation of B cells, or even of T cells, bydirectly binding to the β-subunit of T cell receptors in a non-specificfashion. Another explanation deduces autoimmune diseases from a“Molecular Mimicry”: an exogenous antigen may share structuralsimilarities with certain host antigens; thus, any antibody producedagainst this antigen (which mimics the self-antigens) can also, intheory, bind to the host antigens and amplify the immune response.Autoimmune diseases based on molecular mimicry are known to a skilledperson for various viral and bacterial antigens. The most striking formof molecular mimicry is observed in Group A beta-haemolyticstreptococci, which shares antigens with human myocardium, and isresponsible for the cardiac manifestations of rheumatic fever.

Additionally, according to one further specific aspect, diseases asdefined herein comprise allergies or allergic diseases, i.e. diseasesrelated to allergies. Allergy is a condition that typically involves anabnormal, acquired immunological hypersensitivity to certain foreignantigens or allergens, such as the allergy antigens as defined herein.Such allergy antigens or allergens may be selected from allergy antigensas defined herein antigens derived from different sources, e.g. fromanimals, plants, fungi, bacteria, etc. Allergens in this context includee.g. danders, grasses, pollens, molds, drugs, or numerous environmentaltriggers, etc. Allergies normally result in a local or systemicinflammatory response to these antigens or allergens and lead toimmunity in the body against these allergens. Without being bound totheory, several different disease mechanisms are supposed to be involvedin the development of allergies. According to a classification scheme byP. Gell and R. Coombs the word “allergy” was restricted to type Ihypersensitivities, which are caused by the classical IgE mechanism.Type I hypersensitivity is characterised by excessive activation of mastcells and basophils by IgE, resulting in a systemic inflammatoryresponse that can result in symptoms as benign as a runny nose, tolife-threatening anaphylactic shock and death. Well known types ofallergies include, without being limited thereto, asthma, allergicasthma (leading to swelling of the nasal mucosa), allergicconjunctivitis (leading to redness and itching of the conjunctiva),allergic rhinitis (“hay fever”), anaphylaxis, angiodema, atopy, atopicdermatitis (eczema), urticaria (hives), eosinophilia, respiratory,allergies to insect stings, skin allergies (leading to and includingvarious rashes, such as eczema, hives (urticaria) and (contact)dermatitis), food allergies, allergies to medicine, etc. Treatment ofsuch allergic disorders or diseases may occur preferably bydesensitizing the immune reaction which triggers a specific immuneresponse. Such a desensitizing may be carried out by administering aneffective amount of the allergen or allergic antigen encoded by thenucleic acid as defined herein, preferably, when formulated as apharmaceutical composition, to induce a slight immune reaction. Theamount of the allergen or allergic antigen may then be raised step bystep in subsequent administrations until the immune system of thepatient to be treated tolerates a specific amount of allergen orallergic antigen.

Diseases in the context of the present invention may also include typeII hypersensitivity reactions (cytotoxic, antibody-dependent), includinge.g. autoimmune hemolytic anemia, thrombocytopenia, erythroblastosisfetalis, Goodpasture's syndrome, Graves' disease, Myasthenia Gravis,etc.; type III hypersensitivity reactions (immune complex disease),including e.g. serum sickness, Arthus reaction, Systemic lupuserythematosus (SLE), etc.; type IV hypersensitivity reactions(delayed-type hypersensitivity (DTH), cell-mediated immune memoryresponse, antibody-independent), including e.g. contact dermatitis,Mantoux test, chronic transplant rejection, multiple sclerosis, etc.;and type V hypersensitivity reactions (receptor mediated autoimmunedisease), including e.g. Graves' disease, Myasthenia Gravis, etc.;

In a further preferred embodiment, the inventive vaccine may beformulated as a kit, preferably as a kit of part. Accordingly, thepresent invention also provides kits, particularly kits of parts,comprising the components of the inventive vaccine either alone or incombination with further ingredients as defined above, and optionallytechnical instructions with information on the administration and dosageof the inventive vaccine. The components of the inventive vaccine eitheralone or in combination with further ingredients as defined above may becontained in the kit in either one part of the kit or in different partsof the kit, e.g. each at least one mRNA encoding at least one antigen asdefined above in one part of the kit, and preferably further componentsadmixed to the each at least one mRNA encoding at least one antigen orseparately in a further part of the kit. Such kits, preferably kits ofparts, may be applied, e.g., for any of the above mentioned applicationsor uses.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other, wheresuitable. Furthermore, the term “comprising” shall not be construed asmeaning “consisting of”, if not specifically mentioned. However, in thecontext of the present invention, term “comprising” may be substitutedwith the term “consisting of”, where suitable.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

FIGURES

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1 A: shows the development of the weight of the mice in theexperiment. As a result newborn mice vaccinated with mRNA coding for PR8H1 Hemagglutinin exhibited a significantly better survival (all micesurvived) against influenza challenge infection with control mRNA only(all mice died about 5 days subsequent to vaccination with control mRNAencoding chicken ovalbumin, when vaccinated with control mRNA at thefirst day 8 weeks and died about 6 days subsequent to vaccination withcontrol mRNA, when vaccinated with 8 weeks). Most surprisingly, thesurvival rate was comparable to that of adult mice.

FIGS. 1 B, C show the coding sequence of the mRNAs used for vaccinationof newborn and 8 weeks old mice (see FIG. 1A) coding for PR8 H1 HA(Hemagglutinin of influenza virus A/Puerto Rico/8/1934) (SEQ ID NO: 384)(FIG. 1B) or for Gallus gallus ovalbumine as a control (control mRNA)(SEQ ID NO: 385) (FIG. 1C)

EXAMPLES

The following examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

Example 1—Preparation of mRNA Constructs

For the present examples DNA sequences, encoding PR8 H1 HA(Haemagglutinin of A/Puerto Rico/8/1934) (SEQ ID NO: 384), and Gallusgallus ovalbumine, respectively, as a control (control mRNA) (SEQ ID NO:385), were prepared and used for subsequent in vitro transcriptionreactions.

According to a first preparation, the DNA sequence termed PR8 H1 HA(Haemagglutinin of A/Puerto Rico/8/1934) (SEQ ID NO: 384) (see FIG. 1B)was prepared by modifying the wildtype Haemagglutinin encoding DNAsequence by introducing a GC-optimized sequence for a better codon usageand stabilization. In SEQ ID NO: 384 (see FIG. 1B) the sequence of thecorresponding mRNA is shown. The sequence was furthermore introducedinto a pCV19 vector and modified to comprise stabilizing sequencesderived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)), astretch of 70×adenosine at the 3′-terminal end (poly-A-tail) and astretch of 30×cytosine at the 3′-terminal end (poly-C-tail). Thesequence of the final DNA construct was termed “PR8 H1 HA”.

According to a second preparation, the DNA sequence termed Gallus gallusovalbumine, respectively, as a control (control mRNA) (SEQ ID NO: 385)(see FIG. 1C) was prepared by modifying the wildtype Gallus gallusovalbumine encoding DNA sequence by introducing a GC-optimized sequencefor a better codon usage and stabilization. In SEQ ID NO: 385 (see FIG.1C) the sequence of the corresponding mRNA is shown. The sequence wasfurthermore introduced into a pCV19 vector and modified to comprisestabilizing sequences derived from alpha-globin-3′-UTR (muag (mutatedalpha-globin-3′-UTR)), a stretch of 70×adenosine at the 3′-terminal end(poly-A-tail) and a stretch of 30×cytosine at the 3′-terminal end(poly-C-tail). The sequence of the final DNA construct was termed“Gallus gallus ovalbumine”.

In a further step, the respective DNA plasmids prepared above weretranscribed into mRNA in vitro using T7-Polymerase. Subsequently theobtained mRNA was purified using PUREMESSENGER® (CureVac, Tubingen,Germany).

All obtained mRNAs used herein were furthermore complexed with protamineprior to use. The RNA complexation consisted of a mixture of 50% freemRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1.First, mRNA was complexed with protamine by slow addition ofprotamine-Ringer's lactate solution to mRNA. As soon as the complexeswere stably generated, free mRNA was added, stirred shortly and thefinal concentration of the vaccine was adjusted with Ringer's lactatesolution.

Example 2—Vaccination of Newborn and 8 Weeks Old Mice

In this experiment newborn or 8 weeks old mice were vaccinated twiceintradermally with 80 μg mRNA coding for PR8 H1 HA (Hemagglutinin ofA/Puerto Rico/8/1934; FIG. 1B) or with mRNA coding for Gallus gallusovalbumine as a control (control mRNA; FIG. 1C). The first injection wascarried out at the first day of life (≤24 h) and with 8 weeks,respectively. 5 weeks after the last vaccination the mice werechallenged with a 10fold median lethal dose of PR8 virus (10 LD50). Theweight of the mice was controlled over 2 weeks and the mice were killedwhen they have lost more than 25% of their original weight. The resultsare shown in FIG. 1A. FIG. 1A shows the development of the weight of themice in the experiment. As a result mice vaccinated with mRNA coding forPR8 H1 Hemagglutinin exhibited a significantly better survival (all micesurvived) against influenza challenge infection with control mRNA only(all mice in the control experiment died about 5 days subsequent tovaccination with control mRNA encoding chicken ovalbumin, whenvaccinated with control mRNA at the first day and died about 6 dayssubsequent to vaccination with control mRNA, when vaccinated with 8weeks). All vaccinated newborn mice survived antigen challenge with PR8H1 Hemagglutinin in contrast to the control.

The invention claimed is:
 1. A method for stimulating a protectiveantigen specific immune response in an infant subject comprising,administering to the subject a first effective amount of a compositioncomprising a purified mRNA encoding a viral antigen, said mRNAcomprising a 5′ cap and a polyA tail, wherein said mRNA is complexedwith a cationic or polycationic compound, wherein the subject is aninfant subject being no more than 2 years of age and wherein thecomposition is administered by injection and wherein the viral antigenis an influenza hemagglutinin (HA) virus antigen, or an antigenicfragment thereof.
 2. The method of claim 1, wherein the subject is about3 months to 2 years of age.
 3. The method of claim 2, wherein thesubject is no more than 1 year of age.
 4. The method of claim 3, whereinthe subject is no more than 6 months of age.
 5. The method of claim 1,wherein the composition is administered by intradermal or intramuscularinjection.
 6. The method of claim 5, wherein the composition isadministered by intramuscular injection.
 7. The method of claim 1,wherein the cationic or polycationic compound comprises a cationiclipid.
 8. The method of claim 1, wherein the mRNA comprises a backbonemodification, a sugar modification, or a base modification.
 9. Themethod of claim 8, wherein the mRNA comprises a base modification. 10.The method of claim 1, further comprising administering an adjuvant tothe subject.
 11. The method of claim 10, wherein the adjuvant is animmunostimulatory RNA (isRNA).
 12. The method of claim 1, wherein theantigen-specific immune response comprises production ofantigen-specific antibodies.
 13. The method of claim 12, wherein theantigen-specific immune response comprises an antigen-specific Th1immune response.
 14. The method of claim 1, wherein the compositionfurther comprises a mRNA encoding at least a second viral antigen or anantigenic fragment thereof.
 15. The method of claim 1, furthercomprising administering at least a second dose of the composition tothe subject.
 16. The method of claim 15, wherein the second dose of thecomposition is administered at least 10 days after the firstadministration.
 17. The method of claim 1, wherein the compositionfurther comprises polyethyleneglycol.
 18. A method for stimulating aprotective antigen specific immune response in an infant subjectcomprising, administering to the subject an effective amount of acomposition comprising a purified mRNA encoding, a viral antigen, saidmRNA comprising: a 5′ cap, a polyA tail, and at least one basemodification, said composition comprising a cationic lipid, wherein thesubject is an infant subject being no more than 2 years of age andwherein the composition is administered by intramuscular injectionwherein the viral antigen is an influenza hemagglutinin (HA) virusantigen, or an antigenic fragment thereof.
 19. The method of claim 1,wherein the G/C content of the mRNA encoding the viral antigen isincreased compared to an original RNA sequence encoding the antigen. 20.The method of claim 19, wherein the G/C content of the mRNA encoding theviral antigen is increased by at least 15% compared to an original RNAsequence encoding the antigen.
 21. The method of claim 1, wherein thecationic or polycationic compound is a polypeptide.
 22. The method ofclaim 21, wherein the polypeptide is protamine.
 23. The method of claim1, wherein the viral antigen is full length HA.
 24. The method of claim18, wherein the viral antigen is a full length HA virus antigen.
 25. Themethod of claim 18, wherein the G/C content of the mRNA encoding theviral antigen is increased compared to an original RNA sequence encodingthe antigen.
 26. The method of claim 25, wherein the G/C content of themRNA encoding the viral antigen is increased by at least 15% compared toan original RNA sequence encoding the antigen.
 27. The method of claim18, wherein the subject is about 3 months to 2 years of age.
 28. Themethod of claim 27, wherein the subject is no more than 1 year of age.29. The method of claim 18, wherein the subject is no more than 6 monthsof age.
 30. The method of claim 27, wherein the composition furthercomprises polyethyleneglycol.
 31. The method of claim 18, wherein thecomposition further comprises a mRNA encoding at least a second viralantigen or an antigenic fragment thereof.